Live-Cell Analysis Handbook

A guide to real-time live-cell imaging and analysis Second Edition

Table of Contents

1. Introduction...........................................................................................................................................................................3 2. Cell Culture Quality Control Assays...............................................................................................................................5 3. Kinetic Cell Health and Viability Assays......................................................................................................................9 a. Kinetic Proliferation Assays..........................................................................................................................................12 b. Kinetic Caspase-3/7 and Annexin Apoptosis Assays...............................................................................................21 c. Kinetic Cytotoxicity Assays...........................................................................................................................................29 4. Kinetic Cell Migration and Invasion Assays..............................................................................................................37 a. Kinetic Scratch Wound...................................................................................................................................................40 b. Kinetic Chemotaxis Assays............................................................................................................................................45 c. Kinetic Transendothelial Migration Assays................................................................................................................51 5. Kinetic Assays for Quantifying Protein Dynamics..................................................................................................55 a. Kinetic Antibody Internalization Assays....................................................................................................................57 b. Live-Cell Immunocytochemistry..................................................................................................................................64 6. Kinetic Assays Utilizing Complex Models...................................................................................................................71 a. Kinetic Multi-Spheroid Assays......................................................................................................................................74 b. Kinetic Single Spheroid Assays.....................................................................................................................................81 7. Appendix...............................................................................................................................................................................89 a. Cell Health Protocols i. IncuCyte® Cell Proliferation Assay Protocol....................................................................................................91 ii. IncuCyte® Transduction Protocol.......................................................................................................................94 iii. IncuCyte® Apoptosis Assay Protocol..................................................................................................................97 iv. IncuCyte® Cytotoxicity Assay........................................................................................................................... 100 v. IncuCyte® Immune Cell Killing Assay............................................................................................................. 103 b. Cell Migration and Invasion Protocols i. IncuCyte® Scratch Wound Assay...................................................................................................................... 107 ii. IncuCyte® Chemotaxis Cell Migration Assay................................................................................................. 112 iii. IncuCyte® Chemotaxis Cell Invasion Assay.................................................................................................... 116 iv. Chemotactic Transendothelial Migration Assay........................................................................................... 119 c. Protein Dynamics Protocols i. IncuCyte® Antibody Internalization Assay..................................................................................................... 122 ii. IncuCyte® Live-cell Immunocytochemistry.................................................................................................... 125 d. Spheroid Protocols i. IncuCyte® S3 Multi-Spheroid Assay................................................................................................................. 130 ii. IncuCyte® S3 Single Spheroid Assay................................................................................................................ 133 1

Live-Cell Analysis Handbook â&#x20AC;&#x201D; Second Edition

Introducing Real-Time Live-Cell Analysis

The biomedical world has come a long way since Anton van Leeuwenhoek first observed living cells with a basic microscope in 1674. Using fluorescent probes and modern high resolution imaging techniques it is now possible to view labeled sub-cellular structures at the 10-50 nanometer scale. For researchers working with fixed (dead) cells, organelles can be studied at even higher resolution using electron microscopy. These methods provide tremendous insight into the structure and function of cells down to the molecular and atomic level. The further development of cell imaging techniques has largely focused on resolving greater spatial detail within cells. Examples include higher magnification, three dimensional viewing and enhanced penetration into deep structures. Significant attention has also been paid to temporal resolution – time-lapse imaging has evolved for high-frame rate image capture from living cells to address “fast” biology such as synaptic transmission and muscle contractility. Any consideration for technology advances at lower spatial or temporal detail may initially seem mundane, or even unnecessary. However, this would fail to recognize some key unmet user needs. First, there is an increasing realization that many important biological changes occur over far longer time periods than current imaging solutions enable. For example, maturation and differentiation of stem cells can take hours, days and sometime weeks, which is hard to track using existing methods. Second, imaging techniques are not readily accessible to all researchers nor on an everyday basis. This lack of accessibility is either due to virtue of instrumentation that is expensive and use-saturated or by complex software that renders image acquisition and analysis the sole domain of the expert user. Third, and particularly with regard to time-lapse measurement, the throughput of current solutions is typically too low for frontline use in industrial applications. Finally and most importantly, researchers are increasingly aware that any perturbance of the cells in the process of imaging (e.g. fixing, loss of environmental control) can introduce unwanted and misleading experimental artefacts. Together, these factors frame up the requirement for solutions that enable longer-term, non-perturbing analyses of cells at a throughput and ease of use commensurate with non-specialist users, and at industrial scale. A new generation of specialized compact microscopes and live-cell

imaging devices, are now emerging to meet this need. Designed to reside within the controlled, stable environment of a cell incubator, these systems gather cell images (phase contrast, bright-field and/ or fluorescence) from assay micro-plates automatically, repeatedly and around the clock. Image acquisition is completely noninvasive and non-perturbing to cells, opening up the opportunity to capture the full, and as needed, long-term time course of the biology. Acquisition scheduling, analysis and data viewing can be conducted easily and remotely, without in-depth knowledge of image processing. Data is analyzed on the fly, image by image, to provide real-time insight into cell behavior. We refer to this paradigm, which is differentiated from straight live-cell imaging by the provision of analysed data at scale as opposed to simply images, as ‘real-time live-cell analysis’. While traditional compact microscopes typically only image from a single micro-plate or flask at a time, new live-cell analysis devices such as IncuCyte can automatically capture and analyze images from multiple micro-plates in parallel, thereby significantly increasing throughput (e.g. IncuCyte = 6 x 384 well plates). With the IncuCyte® system, a unique moving optical path design means that the cells and cell plates remain stationary throughout the entire experiment. This further minimizes cell perturbance and enables imaging and analyses of both adherent and non-adherent cell types. This combination of functionality, throughput and ease of use revolutionizes the way researchers can think about imaging assays in living cells. Real time live-cell analysis has now been applied to a wide range of phenotypic cellular assays including cell proliferation, cell death and apoptosis, immune-cell killing, migration, chemotaxis, angiogenesis, neurite outgrowth and phagocytosis. In each case, the full time-course data and ‘minimovies’ of the assay provide greater biological insight than end point assays. Novel analyses such as area under curve, time to signal onset or threshold, and rate parameters (dx/dt) are at times highly value adding. Simply calculating the assay signal at its peak timepoint and/or at the optimal signal/background all helps in assembling robust and reproducible assays. Of course, transient effects of treatments can be detected by kinetic imaging that may otherwise be missed with end-point reads. Due to its non-invasive nature, measurements from cells can be made not only during the assay itself but also during the cell

Live-Cell Analysis Handbook — Second Edition preparation and ‘pre-assay’ stage. For example, the morphology and proliferation rates of cells can be monitored throughout the cell culture period and immediately post-seeding on the microtiter assay plate. The parameter/phenotype of interest can be measured prior to the addition of treatments to provide a within well baseline measure. Quality control of cells and assay plates in this way helps improve assay performance and consistency by ensuring that experiments are only conducted on healthy, evenly plated cultures with the expected cell morphology. The real-time live-cell analysis approach also provides the opportunity to make data driven decisions while the experiment is in progress. A researcher studying the biology of vascular or neuronal networks, for example, may wish to first establish a stable network before assessing the effects of compound treatments or genetic manipulations (e.g. siRNAs). With continuous live-cell analysis, it is straightforward to temporally track network parameters and use the real time data to judge when best to initiate the treatment regimes. The timing of adjunct studies such as analysis of metabolites or secreted proteins in supernatants can also be guided. Drug washout studies may be performed using the real time data to identify when an equilibrium response occurs and to trigger the timing of the washout regime. If for any reason it transpires that the experiment is not performing as expected then treatments could be withheld to save expensive reagents and follow-on experiments can be initiated more quickly to make up time.

Real-time live-cell analysis is extremely helpful when developing, validating and troubleshooting phenotypic assays. Within a small number of assay plates it’s usually possible to obtain a clear understanding of the relationship over time between assay signal and treatments, cell plating densities, plate coatings and other protocol parameters. Scrutiny of the kinetic data and ‘mini-movies’ from each well help to rapidly pinpoint sources of within- and across-plate variance and to validate the biology of interest. This is particularly true for more advanced cell systems such as co-cultures where far more permutations and combinations of protocol parameters exist (e.g. cell plating ratios) and the biology is more complex. In summary, real-time live-cell analysis is re-defining the possibilities and workflows of cell biology. The combination of ease of use, throughput, long term stability and non-invasive measurement enables researchers to monitor and measure cell behaviors at a scale and in ways that were previously not possible, or at the least, highly impractical. In the following chapters of this handbook, we illustrate this with a range of different application examples.

Cell Culture Quality Control Assays Real-time monitoring and analysis of cell culture conditions and techniques

A large number of variables exist that alter the growth and function of cells in culture. Many sources of variability are largely uncontrollable because they are inherent to the stochastic processes in biological systems. Other factors can be identified and controlled. Some key controllable factors include: •• Poor CO2 incubator performance due to lack of calibration and stability of temperature, humidity and CO2 over time. •• Non-quantitative and/or inconsistent procedures for feeding and splitting cell cultures prior to running cell-based assays including inconsistent limits of cell density and feeding schedules, inconsistent cell density at the time of assay and changes to cell morphology. •• Alterations in media components due to lot-to-lot differences and alteration of concentrations over time due to degradation. •• Differences in cell culture growth surfaces such as variability among suppliers, vessel surface treatments and lot-to-lot variability. •• Biological issues resulting from growing cells in continuous culture for extended periods of time which in turn increases the risk of phenotypic drift, contamination by infectious agents and cross contamination with other cell types.

Each of these controllable variables can adversely affect the results and interpretation of data obtained from downstream assays leading to lost time and a waste of valuable, and at times, costly reagents. A key element in controlling adverse variables is to standardize on objective metrics, thereby eliminating human subjectivity and interpretation.

Advantages of performing quality control of cell cultures via continuous monitoring The IncuCyte® real-time live-cell analysis system provides a label-free, non-invasive method for monitoring cells directly in the incubator. In sharp contrast to manual monitoring of cell culture processes, quality control monitoring with real-time livecell analysis automates data capture and cell assessment (Figure 1). Cells can be monitored around-the-clock and at precise, regularly scheduled sampling intervals. In addition to supporting decisions for current culture processes, historical information can be retrieved months or years later for comparison of cell lines and culture growth characteristics.

Manual Cell Culture Monitoring Initial cell density— subjective

Seed vessel Initial cell density— measured for every well

Live-Cell Analysis

Assessments made at random locations with variable frequencies— qualitative and operator dependent

Cell utilization decisions including timing— subjective

Monitor growth

Utilize cells

Assessments made at precise locations with scheduled sampling intervals— objective growth curves and morphology data support decisions

Split, treatment, harvest or assay decisions based on review of both morphology and quantitative confluence measurements

TIME

Figure 1. Comparison of cell culture monitoring methods. Vertical arrows represent interaction points with the cell culture using manual methods and real-time live-cell analysis. Subjective decisions made during manual monitoring result in variability for cell-based assays. 5

Live-Cell Analysis Handbook — Second Edition

Cell Culture Quality Control Assays

The IncuCyte system is compatible with multiple different types of cell culture vessels and can monitor multiple vessels at a time. Data consists of objective, image-based growth metrics to capture transient and time-dependent events.

•• Documentation of cell morphology Changes in cell morphology due to cell seeding density or phenotypic drift can be identified using a range of magnifications to capture fine details of cells and spatial coverage of cell populations.

In order to establish quality cell cultures and improve experimental outcomes in downstream assays, various aspects of cell culture need to be tightly controlled, optimized and documented.

•• Accurate assessment of overall monolayer By imaging multiple areas of your vessel or using a whole-well imaging mode, spatial variations in cell distribution can be quantified and then reduced.

•• Growth conditions Prior to utilizing cells in quantitative assays, it is important to optimize and define cell culture regimens. Documenting variations in cell growth due to factors such as; lot-to-lot differences in cell culture media, changes in media component concentrations over time due to degradation, and inconsistent feeding schedules can all be identified using label-free phase image segmentation techniques that generate confluence measurements.

•• Cell seeding densities To improve assay quality and consistency of kinetic assays, it is important to minimize variations in cell seeding densities due to pipetting errors during plate seeding which result in differential growth rates. Measuring confluence before utilizing assay plates eliminates assay variation, resulting in reproducible conditions.

Figure 3 shows results of a study to assess optimal levels of fetal bovine serum (FBS) and cell density using the IncuCyte system. Live-cell analysis can be used to accurately mask different morphologies and quantify label-free proliferation over time, as shown in Figure 4 below. In 4A and 4B, the variation in cell seeding density-dependent proliferation in two different cell lines, A172 human glioblastoma and LNCap human prostate carcinoma, is exemplified. Understanding cell seeding variation and its effects on proliferation is important for controlling assay variability. As shown in 4C, images reveal accurate segmentation of cell types irrespective of morphology.

•• Documenting time-dependent variables Assessment of cell behavior and growth in association with cell treatment times can define experimental conditions to ensure consistent results.

Cell Culture QC Assays Measuring proliferation to optimize growth conditions and seeding densities

Figure 3. Human umbilical vein endothelial cells (HUVECs) cultured in basal cell media containing growth factors and supplemented with decreasing amounts of FBS. The optimal concentration of FBS for normal propagation was determined to be >2.5%.

The IncuCyte system can be used to track cell proliferation over time using label-free phase segmentation (confluence) metrics (Figure 2), allowing for the assessment of cell growth as well as monitoring cell culture and assay optimization.

B C

Figure 2. T-flask vessel view shows location of images acquired (A). Proliferation time-course for HT-1080 human fibrosarcoma cells is shown (B). (C) HD-phase time-lapse images of HT-1080 cells (confluence mask overlaid; right panels). 6

Figure 4. Quantification of A172 human glioblastoma (A) and LNCap human prostate carcinoma (B) growth curves using label-free confluence analysis. (C) HD-phase contrast images of A549 human lung carcinoma, HT-1080 human fibrosarcoma and LNCap cells shown without and with confluence mask (orange). Images reveal accurate confluence masking of all three cell types of varied morphology.

Live-Cell Analysis Handbook â&#x20AC;&#x201D; Second Edition A

Figure 5. Images (A) and data (B) illustrating the effect of pipetting errors on growth. Wells were seeded at the same cell density and then checked prior to an endpoint assay for growth. Initial confluence for four wells were found to be ~50% that of the median initial confluence for the plate, a variable likely caused by pipetting errors.

Conclusions With real-time live-cell analysis it is possible to follow and quantify cell growth over time, effectively revealing both transient and time-dependent phenomena. This type of analysis is a powerful tool for cell culture quality control, providing a quantitative, objective, non-invasive and kinetic method of analyzing living cells, unperturbed in the incubator. Use of the IncuCyte system to document and monitor routine cell culture can improve cell-based assay quality and consistency.

Figure 6. T-25 flask exhibiting uneven growth, often related to poor culture technique. Two representative images from a single flask show differences in confluence. 8

Kinetic Cell Health and Viability Assays Real-time automated measurements of cell health and viability

Measurements of cell health are essential for studying the effects of drugs, culture conditions or genetic modifications on cell growth or viability. Such studies are used to rank compounds in drug discovery screens, identifying off-target toxic compounds, as well as to investigate the cellular changes that underline disease pathologies.1 In order to assess cell health and viability, a variety of end-point assays have been employed, such as ATP assays, LDH assays and vital dyes used in flow cytometry, but all fail to take into account the kinetics or have the capability to make simultaneous measurements in a single well. Understanding the biological time-dependence, along with the ability to evaluate multiple methods by which a cell dies, offers considerable advantages in the characterization of test compounds.2 IncuCyte® cell health and viability assays allow for the kinetic evaluation of effective concentration of the compound and the time needed for the compound to perturb the target, thereby discriminating if the test agent is fast acting or prolonged. Another advantage is the ability to perform multi-parametric analysis using non-perturbing reagents within a single well, enabling detection of the mechanism of cell death as well as monitoring cell viability utilizing reagents that label cell nuclei. These image-based detection methods can all be qualitatively validated with images to evaluate morphological changes associated with cell death.

Live-cell imaging and analysis approaches The IncuCyte cell health and viability assays enable the detection and analysis of cell proliferation, viability, apoptosis, and cytotoxicity. Images of cell cultures in 96- or 384-well formats are automatically acquired and analyzed to generate time courses and reveal concentration-dependent responses that can be used to calculate EC50 or IC50 values. Proliferation and viability assays •• Measure growth or growth inhibition in real time using label-free confluence measurements or counts of cells with fluorescently-labeled nuclei. Apoptosis assays •• Detect apoptosis in living cells and in real time using mixand-read reagents that measure caspase-3/7 activity or phosphatidyl serine externalization. Cytotoxicity assays •• Measure cell death upon loss of plasma membrane integrity over time using simple mix-and-read protocols.

Furthermore, these advantages of live-cell analysis are also ideally suited to meet the needs of more dynamic cellular models, such as co-culture immune cell killing assays, which require robust temporal and spatial quantitative measurements in physiologicallyrelevant conditions. Continuous live-cell imaging makes it possible to measure and visually validate the complex and dynamic interactions between target and effector cells in co-culture for the reliable analysis of new potential immunotherapies.

Live-Cell Analysis Handbook — Second Edition

Kinetic Cell Health and Viability Assays

How live-cell health and viability assays work IncuCyte cell health reagents (Caspase-3/7 Apoptosis Reagent, Annexin V Red or Green, and Cytotox Red or Green) are added directly to tissue culture growth medium using simple mix andread protocols that do not require washing, fixing or lifting. Images are then automatically acquired and analyzed in 96- or 384-well microplate format to elucidate concentration and time dependent effects of treatments.

In addition, IncuCyte® NucLight Red or Green lentivirus reagents label cell nuclei without affecting cell growth or morphology and provide a means to kinetically quantify cell proliferation over time. In combination, IncuCyte cell health reagents and NucLight labels provide a multiplexed way to differentiate inhibition of cell growth and induction of cell death.

References 1. 2.

Kepp, O., Galluzzi, L., Lipinski, M., Uan, J. and Kroemer, G. Cell death assays for drug discovery. Nature Reviews 2011 10:221. Abassi, Y.A., Xi, B, Zhang, W., Ye, P., Kirstein, S.L, Gayloard, M.R., Feinstein, S.C., Wang, X. and Xu, X. Kinetic Cell-Based Morphological Screening: Predisction of Mechanism of Compound Action and Off-target Effects. Chemistry & Biology 2009 16:712.

Recent IncuCyte Publications Balvers, et. al., investigated the therapeutic benefit of combination therapy in patient-derived glioma stem-like cells (GSC). The current standard of care for Glioblastoma Multiforme consists of radiation along with temozolomide (TMZ) chemotherapy. A possible strategy to increase the efficacy of TMZ is through interference with the DNA damage repair machinery, by poly(ADP-ribose) polymerase protein inhibition(PARPi). Proliferation assays consisted of longitudinal imaging of cell confluency in the IncuCyte system. Phase contrast images were acquired at 1-3hr intervals and confluence per well was calculated. ABT-888 enhances cytotoxic effects of temozolomide independent of MGMT status in serum free cultured glioma cells. Balvers, R. K., Lamfers, M., Kloezeman, J. J., Kleijn, A., Berghauser Pont, L., Dirven, C. and Leenstra, S. Journal of Translational Medicine 2015 13:74.

Ong, et. al., evaluated PAK1 dysregulation (copy number gain, mRNA and protein expression) in breast cancer tissues. A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. The IncuCyte system was used for caspase 3/7 activation apoptosis assays. Cells were treated with DMSO, FRAX1036, and/or docetaxel. Caspase 3/7 reagent was added and cells imaged every 2 hours or 4 hours for 36 to 72 hours. Data was analyzed using IncuCyte analysis software to detect and quantify apoptotic cells.

Skolekova, et. al., measured caspase-3/7 activity corresponding to the induction of apoptosis in human mesenchymal stromal cells (MSCs) cultivated in the presence of cisplatin using the IncuCyte platform. Kinetic activation of caspase-3/7 was monitored and quantified using the IncuCyte® FLR object counting algorithm. The authors showed that the secretory phenotype and behavior of mesenchymal stromal cells influenced by cisplatin differed from naïve MSC. MSC were more resistant to cisplatin, which was cytotoxic for tumor cells. Cisplatin-induced mesenchymal stromal cells-mediated mechanism contributing to decreased antitumor effect in breast cancer cells. Skolekova, S., Matuskova. M., Bohac, M., Toro, L., Durinikova, E., Tyciakova, S., Demkova, L., Gursky, J., and Kucerova, L. Cell Communication and Signaling. 2016 14:4.

Greenberg, et. al., studied whether Bcl-2–IP3R interaction is a potentially useful therapeutic target in small cell lung carcinoma (SCLC). The IncuCyte platform was used to detect caspase activation and nuclear condensation in SCLC cell lines. Synergistic killing of human small cell lung cancer cells by the Bcl-2-inositol 1,4,5-trisphosphate receptor disruptor BIRD-2 and the BH3-mimetic ABT-263. Greenberg, E. F., McColl, K. S., Zhong, F., Wildey, G., Dowlati, A., Distelhorst, C. W. Cell Death and Disease 2015 6:e2034.

Yang, et. al., describe characterization of a quadruplex dye (G4-REP; quadruplex-specific red-edge probe) that provides fluorescence responses regardless of the excitation wavelength and modality thus allowing for diverse applications and most imaging facilities. Authors demonstrated use via cell images and associated quantifications collected through use of the IncuCyte real-time live-cell imaging system over an extended period, monitoring both non-cancerous and cancerous human cell lines. Real-time and quantitative fluorescent live-cell imaging with quadruplex-specific red-edge probe (G4-REP). Yang, S. Y., Amor, S., Laguerre, A., Wong, J. M. Y., Monchaud, D. Biochimica et Biophysica Acta (BBA) - General Subjects http://dx.doi.org/10.1016/j.bbagen.2016.11.046.

Ano, et. al. sought to elucidate specific compounds that strongly suppress microglial inflammation by screening dairy products fermented with Penicillium candidum. Quantitative live cell imaging to assess apoptosis or neuronal cell-death was performed using an IncuCyte real-time imaging system. Images were obtained every 3 hour at 20x magnification in phase contrast mode. The number of cells that had undergone apoptosis or cell-death was determined using the object counting algorithm. Identification of a novel dehydroergosterol enhancing microglial anti-inflammato ry activity in a dairy product Fermented with Penicillium candidum. Ano, Y., Kutsukaka, T., Hoshi, A., Yoshida,, A., Nakayama, H. PLoS On 10, e0116598, 201.

Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents. Ong, C. C., Gierke, S., Pitt, C., Sagolla M., Cheng, C. K., Zhou, W., Jubb, A. M., Strickland, L., Schmidt, M., Duron, S. G., Campbell, D. A., Zheng, W., Dehdashti, S., Shen M., Yang, N., Behnke, M. L., Huang W., McKew, J. C., Chernoff J., Forrest, W.F., Haverty, P. M., Chin S., Rakha, E. A., Green, A. R., Ellis, I. O., Caldas, C., O’Brien, T., Friedman, L. S., Koeppen, H., Rudolph, J., Hoeflich, K. P. Breast Cancer Research 2015 17:59.

Barsyte-Lovejoy, et. al. provide a protocol for measuring early toxicity/apoptosis response to chemical probes using the IncuCyte platform. Methods in Enzymology, 2016. Chapter 4: Chemical Biology Approaches for Characterization of Epigenetic Regulators. Barsyte-Lovejoy, D., Szewczyk, M.M., Prinos, P.

Novotny, et. al. evaluated the ability of reversible HER2 inhibitors to inhibit signaling and proliferation in cancer cell lines driven by HER2– HER3 heterodimers activated via various oncogenic mechanisms. The IncuCyte platform was used for real-time cell proliferation assays. CHL-1 cells were plated in clear-bottom black 96-well plates (Corning; 3904) and allowed to adhere overnight. The following day, media was changed to fresh media that contained either DMSO or compound. Confluence was measured every 2 h for 96 h using two bright-field images per well. Overcoming resistance to HER2 inhibitors through state-specific kinase binding. Novotny, C.J., Pollari, S., Park, J.H., Lemmon, M.A., Shen, W., Shokat, K.M. Nature Chemical Biology 2016 12:923.

Live-Cell Analysis Handbook — Second Edition

Kinetic Proliferation Assays

IncuCyte® Proliferation Assays at a Glance

Kinetic Proliferation Assays

IncuCyte® live-cell imaging enables non-invasive, label-free and fully kinetic measurements of cell growth based on area (confluence) metrics generated from high quality phase images, which remains a high value and valid approach for many applications (Figure 1). Additionally, continuous live cell assays for non-adherent cells are possible and offer a simple methodology which does not interfere with cells’ inherent biology, as the cells stay stationary inside a standard tissue culture incubator while IncuCyte® optics move.

Powerful solutions for accurate and reproducible cell proliferation measurements

Introduction Cell proliferation assays are a cornerstone of cancer therapeutic, developmental biology, and drug safety research. Analysis of the sustained signaling pathways that underlie the progression of tumors, for example, accounts for >10,000 manuscripts in PubMed, the majority of which use cell proliferation analysis to evaluate tumor cell growth. Despite this, there has not been a direct, straightforward, industrialized method for quantifying cell proliferation as a continuous event. Rather, the majority of measures are endpoints or at best a series of concatenated endpoints to measure the time-course. Many biochemical detection measures (e.g. LDH, ATP detection) are indirect and subject to artifacts that cannot be readily verified by morphology changes. Antibody- and chemical probe-based high content imaging methods address this limitation but are not amenable to long term temporal assays. Challenges in monitoring cell proliferation via traditional single point non-image based techniques include: •• Use of concatenated endpoints to measure proliferation over time utilizes samples from different wells, potentially introducing artifacts.

Image-based analysis alleviates these associated challenges by allowing for non-invasive, repeated scanning of the same sample over time, either using label-free transmitted light image acquisition or with the use of reagents that fluorescently label cell nuclei without affecting the growth or morphology of the cell. The ability to label cells using non-perturbing reagents also allows for the kinetic analysis of multiple cell types in co-culture, a model often demonstrated to be more representative of in vivo biology. Additionally, live-cell analysis using the IncuCyte system is compatible with non-adherent cells, as cell perturbance is minimized by moving the optics, while the cells stay stationary. This is in contrast to traditional image-based analysis via microscopy, where optics are stationary and samples undergo movement, making quantitation over time difficult.

To resolve the challenge of cells with very low contrast, or the lack of linearity between confluence and actual cell number, the IncuCyte system can be used to measure kinetic cell proliferation based on direct cell (nuclear) counting using novel nuclearrestricted green or red fluorescent proteins, NucLight Green and NucLight Red. This integrated approach enables a mediumthroughput, content-rich, fully kinetic solution.

IncuCyte® NucLight lentivirus reagents enable efficient, nonperturbing, nuclear labelling of living mammalian cells. They are compatible with convenient transduction protocols and enable real-time cell counting and the calculation of cell doubling times. IncuCyte NucLight lentivirus reagents provide homogenous expression of a nuclear-restricted GFP (green fluorescent protein) or mKate2 (red fluorescent protein) in your choice of primary, immortalized, dividing, or non-dividing cells without altering cell function and with minimal toxicity. These reagents are ideal for generating stable cell populations or clones using puromycin or bleomycin selection.

In this chapter, we will illustrate how image-based analysis allows for the kinetic quantification of cell proliferation without disturbing the cells being monitored. This is especially valuable for complex co-culture models as well as monitoring nonadherent cells.

•• Many detection reagents perturb cell growth and morphology and are therefore unsuitable for kinetic analysis. •• Growth rates of cells grown in co-cultures can not be distinguished.

Figure 1. Proliferation is successfully measured using an IncuCyte phase-only processing module. An IncuCyte mask (orange) analyzes a phasecontrast image of proliferating neuro-2a cells. The proliferation of neuro-2a cells decreases under increasing concentrations of all-trans retinoic acid.

Live-Cell Analysis Handbook — Second Edition

Kinetic Proliferation Assays

Sample Results Quantitative measurement of cancer cell proliferation in a co-culture model

Figure 2. A wide range of co-culture-based assays are possible by fluorescently labeling the nuclei of one or more cell types. (HT-1080 red; A549 green) with a non-perturbing nuclear-targeted fluorescent protein. IncuCyte fluorescent object counting software successfully quantifies real-time cell proliferation.

Certain cancers are resistant to chemotherapy due to the biological activity of their neighboring cells, or within the context of the tumor microenvironment. For example, stromal cells have been observed to rescue tumors from drug-induced toxicity by secreting growth factors that impede apoptotic pathways.1 Such cell interactions may be illuminated by juxtaposing monocultures and co-cultures through in vitro assays. In this study, we examined the culture-dependence of the SK-BR-3 cell line, a type of breast adenocarcinoma cell that overexpresses HER-2. A previous study demonstrated that monocultures of SK-BR-3 cells are sensitive to the drug lapatinib, which induces cell death by inhibiting tyrosine kinase activity of the HER-2 and EGFR pathways.2 However, another study showed that when SK-BR-3 cells were co-cultured with normal skin fibroblasts (CCD-1068Sk), these stromal cells were able to rescue the inhibitory effect of lapatinib. Interestingly, no such rescue effect was observed when SK-BR-3 cells grown in co-culture with normal human mammary fibroblasts (HMF).1 In a similar study, the proliferation of SK-BR-3 cells in monoculture and co-culture were continuously and kinetically monitored in the presence of increasing concentrations of lapatinib. SK-BR-3 cells were quantified using IncuCyte’s nucleus counting algorithm. This method allows real-time

A Shortcomings of Traditional Assays

Live-Cell Imaging and Analysis Approaches

• Assays use end points or concatenated end points to measure time-course.

• Arbitrarily defined end points are not required, enabling data driven decisions while the experiment is in progress.

• Detection methods are often indirect.

• Non-invasive, fully kinetic measure of cell growth in 96- and 384-well microplates.

• Subject to artifacts that cannot be readily verified by morphology changes.

• Measurement of un-labeled cells based on area (confluence) metrics.

• Cells with low contrast can be difficult to quantify. • Relationship between confluence and true cell number becomes non-linear when morphology changes.

High-throughput compound testing is essential for efficiently

• Direct nuclear counting of mono- and co-cultures with novel nuclear-restricted green and red fluorescent proteins. • All data and time points can be verified by inspecting individual images and/or time-lapse movies. • Cell morphology observations provide additional validation and insight into mechanistic differences between treatments or conditions. • Proliferation assays can be used with non-adherent cells and are non-perturbing when using an optical system that moves rather than a motorized X-Y stage.

Figure 3. Proliferation of SK-BR-3 cells in co-culture and monoculture under lapatinib treatment. SK-BR-3 cells expressing a nuclear restricted red protein were grown with normal skin fibroblasts (CCD-1068Sk), human mammary fibroblasts (HMF), or in monoculture, and treated with varying concentrations of lapatinib for 8 days. (A) Nuclear counts per mm2 of SK-BR-3 cells grown with or without stromal cells in the presence of 556 nM lapatinib illustrate the rescue effect of CCD-1068Sk fibroblasts compared to HMFs and mono-culture. (B) Area under the curve of nuclear counts per mm2 over time for each concentration (n=4) was used to calculate and compare IC50 values of SK-BR-3 cells grown with or without stromal cells.

High-throughput compound testing using NucLight Green labeled HT-1080s advancing promising drugs through the drug discovery pipeline. To examine the ability of the IncuCyte system to meet this need, cell proliferation was measured over time in a higher-throughput format. To assess many pharmacological agents simultaneously, sixteen literature-standard compounds (Table 1) were applied to HT-1080 tumor-derived fibrosarcoma in a 384-well format (Figure 4). An 11-point concentration response curve was constructed

cell counting based on nuclear restricted fluorescent protein expression. Kinetic graphs of nuclear counts per mm2 over time show SK-BR-3 cells grown with CCD-1068Sk fibroblasts grow at a significantly higher rate than those grown alone or with HMF’s in the presence of ≈500 nM lapatinib (Figure 3A). In addition, IC50 values (calculated using the area under the curve (AUC) of nuclear counts per mm2 over time) provide quantitative evidence for the differences in drug response between monoculture and co-cultures in the presence of lapatinib. Specifically, SK-BR-3 cells grown with CCD- 1068Sk fibroblasts are the least sensitive to lapatinib with an IC50 value of 1.162 μM, followed by SK-BR-3 cells grown with HMF’s with an IC50 value of 0.581 μM (Figure 3B). Interestingly, SKBR-3 cells grown in mono-culture remain effectively sensitive to lapatinib with an IC50 value of 0.015 μM (Figure 3B) which is comparable to the published IC50 value, 0.037±0.031 μM.2 These striking data very clearly illustrate the difference in SK-BR-3 cell proliferation in the presence of stromal cells, thus highlighting the importance of considering the effect that the tumor microenvironment can have on drug resistance.

for each compound (Figure 5). Of the 16 compounds tested, the rank order of potency for inhibition of cell proliferation was: doxorubicin = staurosporine = camptothecin > mitomycin C > cycloheximide = RITA > PD-98059 > FAK inhibitor 14 = cisplatin > 10-DEBC = Chrysin = Compound 401. TAME, PAC1, KU0063794 and FPA-124 had little or no effect on cell proliferation under the conditions of the experiment.

Live-Cell Analysis Handbook — Second Edition DRUG

DESCRIPTION

Doxorubicin

chemotherapy drug, intercalates DNA3

Camptothesin

alkaloid inhibits topoisomerase, causing DNA damage3

Staurosporine

potent alkaloid inhibitor of protein kinase4

Mitomycin C

chemotherapy drug, alkylates DNA5

Cycloheximide

protein synthesis, inhibitor6

RITA

(reactivation of p53 and induction of tumor cell apoptosis) a small molecule, binds p537

PD-98059

MAPK1/2 inhibitor8

Cisplatin

chemotherapy drug acts through crosslinking DNA9

FAK-inhibitor 14

selective inhibitor of focal adhension kinase10

10DEBC

selective inhibitor of Akt11

Chrysin

a flavonoid observed to inhibit growth in cancer cells12

TAME

tert-Amyl methyl ether; a gasoline additive with suspected toxic effects upon inhalation13

PAC1

(pro-caspase activating compound-1), a small-molecule activator of procaspase-3 to caspase-314

KU0063794

specific inhibitor of mTORC1/215

FPA-124

Akt inhibitor16

Compound 401

inhibitor of DNA-dependent kinase and mTOR17

Kinetic Proliferation Assays

Table 1. Drugs identified in literature as relevant to cell proliferation.

Figure 5. Concentration-response curves for a selected set of literature standard inhibitors of cell proliferation in HT-1080 NucLight Green cells. Abscissa: -log M [compound], ordinate: maximum nuclear count (per mm2). Each point represents the average data from 2 wells from a single 384-well compound plate. The lines of best fit are a 4-parameter logistic equation calculated in GraphPad Prism™.

To verify and extend these findings, representative images and time-lapse videos of the cells exposed to test compounds at selected concentrations were inspected (Figure 6). Following exposure to an IC80 concentration for 24h, staurosporine produced profound changes in cell morphology, with extensive branching and condensation of the nucleus and cell body. The cells lost motility and there was clear evidence of cytotoxicity. In contrast, the inhibition of cell proliferation by RITA (also IC80,

Control

Figure 4. 384-well microplate view of HT-1080 NucLight Green cell proliferation with 16 different compounds, 11-point concentration-response curves in duplicate (different colors, high to low concentrations left to right). Columns 15 and 16 are vehicle (0.5% DMSO) and CHX (3 μM) controls, respectively. Note the potent concentration-dependent inhibition of cell proliferation for certain compounds (e.g. row J, row M, row O), and weaker effects/inactivity of others (e.g. row A, row P). Abscissa: time (0-72h), ordinate: fluorescent object count per well (0-3800). 16

Staurosporin (37 nM)

Camptothecin (412 nM)

24h) was not accompanied by any obvious morphological changes. At anti-proliferative concentrations, both camptothecin and doxorubicin treated cells appeared healthy with no evidence of cell death, suggesting that senescence had occurred. 10-DEBC (11 mM, 24h) produced overt cytotoxicity and complete cell lysis. These data show the potential of this kinetic and morphological approach to the screening, prioritization, and classification of compounds in drug discovery.

RITA (3.7 μM)

Doxorubicin (46 nM)

10-DEBC (11 μM)

Figure 6. Representative ‘blended’ phase contrast/fluorescence images of HT- 1080 NucLight Green cells treated with different test compounds as labeled. Images shown were taken 24 h post compound treatment (10x). Note the reduction in total cell number for each treatment compared to the vehicle control, and the profound differences in cell morphology.

Live-Cell Analysis Handbook — Second Edition

Kinetic Proliferation Assays

Continuous live-cell proliferation, clustering and viability assays for T-cells To investigate the effect of cell density on T-cell proliferation and cluster formation, isolated human peripheral blood mononuclear cells (PBMCs) were seeded at various cell densities (20-50 K/ well) on poly-l-ornithine (PLO) coated flat bottom 96-well plates. Cells were grown in the absence or presence of T-cell activators (a-CD-3 (100 ng/ml), hrIL-2 (10 ng/ml)) and HD phase contrast images were captured on an IncuCyte system over 144 h. Images were analyzed for phase confluence (%) as a measure of cell proliferation and the number of phase objects above an area threshold (cluster count/well) to define cluster formation. The kinetic graph (Figure 7A) demonstrates little or no proliferation

Proliferation

under basal conditions (grey lines) but rapid proliferation in the presence of activators (blue lines), which is seeding cell density dependent. Following activation, T-cells rapidly form clusters, which is displayed as a cluster count increase (Figure 7B) over time. Both data sets show an increase in the rate of proliferation or cluster formation with cell density (bar graphs, Figures 7A and 7B). The confluence in unstimulated PBMCs can be seen to drop over time due the possible presence of phagocytes. The IncuCyte system acquired phase-contrast images of IL-2/anti-CD3 activated PBMCs, confirming cluster formation as illustrated in Figure 7C.

Cluster formation

Additionally, the IncuCyte system was used to study effects of L-kynurenine (L-kyn) on T-cell proliferation and clustering. L-kyn is a metabolite formed from the catabolism of L-tryptophan by the enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). Some cancers increase L-kyn production in

Proliferation

a bid to block antigen-driven T-cell proliferation and induce T-cell death, thus allowing cancer cells to escape immune surveillance. Inhibitors of IDO and/or TDO are therefore promising therapeutic targets for the treatment of cancer. Figure 9 shows time-course results of L-kyn inhibition of T-cell proliferation and clustering.

Cluster formation

C Automated 96-well continuous analysis

Figure 7. T-cell proliferation and clustering is seeding-density dependent. T-cells demonstrate little or no proliferation under basal conditions but rapidly proliferate when activated (e.g. by IL- 2, anti-CD3, anti-CD28). Following activation, T-cells clusters formed after activation can be imaged, enabling quantification of this phenotype.

To capture the stimulus dependent effect on T-cell proliferation and cluster formation, isolated human PBMCs were seeded (20 K/ well) on a PLO-coated flat bottom 96-well plate and grown in the presence of various T-cell activator combinations, hrIL-2 (10 ng/ ml), a-CD-3 (0.1 ng/ml) and/or a-CD28 (1-100 ng/ml). HD phase contrast images were captured on the IncuCyte system over 240 hours and analyzed for phase confluence (%) as a measure of cell proliferation and the number of phase objects above an area threshold (cluster count/well) to define cluster formation. As

Proliferation

illustrated in Figure 8A, little or no cell proliferation was observed in the presence of IL-2 alone, however, moderate proliferation was observed with the addition of a-CD-3. T-cell proliferation was further enhanced by the addition of a-CD-28, revealing a concentration dependent response (Figure 8A). A similar stimulus dependent cluster formation prolife was also measured in the presence of various T-cell activator combinations (Figure 8B). These data show the potential of this kinetic and morphological approach to evaluating immune cell activation.

Cluster formation

Figure 9. L-kynurenine inhibits T-cell proliferation and clustering. Data demonstrates a clear concentration-related inhibition of IL-2/antiCD3 activated T-cell proliferation and clustering with the addition of exogenous L-kyn, over time. Time-course profiles enabled AUC analysis and generation of concentration-response curves from which IC50 values for inhibition of proliferation and clustering were determined.

Conclusions The IncuCyte live-cell analysis system in conjunction with NucLight reagents provides a live cell, kinetic assay for the measurement of proliferation. This strategy has demonstrated quantitative and reproducible analysis of both adherent and non-adherent cells, in monoculture and importantly, co-culture. It also gives the user the ability to monitor morphological changes in parallel with quantification, the combination of which is a powerful and unique tool for detecting pharmacological or genetic manipulations that alter cell viability. •• Label-free confluence measurements can be generated using phase image segmentation; ideal for mono-culture analysis, when using cells lines that are difficult to infect with cell nuclei labels, or when multiplexing with green or red cell health reagents.

Figure 8. T-cell activation is stimulus and concentration-dependent. Data shown are for PBMCs treated with combinations of IL-2, anti-CD3, and anti-CD28.

•• Proliferation assays can be run in microplates (96- well and 384-well) with high precision and reproducibility. In 384well plates, a mix and read assay is exemplified whereby full concentration-response curves of 16 standard anti-proliferative agents were compared. In a single IncuCyte instrument 6 x 384well plates can be monitored providing >2000 wells of parallel data acquisition. •• All data and time points can be verified by inspecting individual images and/or time-lapse movies. Cell morphology observations provide additional validation and insight into mechanistic differences between treatments or conditions. •• Non-adherent cell proliferation and clustering can be visualized due to the non-perturbing nature of the IncuCyte. Nonadherent cells stay stationary as the optics move.

•• Cell proliferation assay can be combined with non-perturbing reagents that label cell nuclei in order to enumerate cell number and for complex-co-culture models. 19

Live-Cell Analysis Handbook â&#x20AC;&#x201D; Second Edition

References 1.

Straussman R. et al.: Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature 2012, 487 (7408):500-504.

Konecny GE, et al. Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastusumabtreated breast cancer cells. Cancer Res 2006, 66(3):1630-1639.

Sappal, DS, et al. Biological characterization of MLN944: A potent DNA binding agent. Molec. Cancer Ther. 2004, 3(1):47.

Seynaeve, CM. et al. Differential inhibition of protein kinase C isozymes by UCN-01, a staurosporine analogue. Mol. Pharmacol. 1994, 45(6):1207-1214.

Palom, Y. et al. Bioreductive metabolism of mitomycin C in EMT6 mouse mammary tumor cells: cytotoxic and non-cytotoxic pathways, leading to different types of DNA adducts. The effect of dicumaro. Biochem. Pharamcol. 2001, 61(12):1517-1529.

Chang, TC et al. Effects of transcription and translation inhbitors on a human gastric carcinoma line. Potential role of Bcl-X(S) in apoptosis triggered by these inhibitors. Biochem. Pharmacol. 1997, 53(7)96-977.

Roh, JL et al. The p53-reactivating small-molecule RITA enhances cisplatin-induced cytotoxicity and apoptosis in head and neck cancer. Cancer Let. 2012, 325(1):35-41.

Aravena, C et al. Potential Role of Sodium-Proton Exchangers in the Low Concentration Arsenic Trioxide-Increased Intracellular pH and Cell Proliferation. PLoS One 2012, 7(12): e51451.

Hu, W. The anticancer drug cisplatin can cross-link the interdomain zinc site on human albumin. Chem. Commun. 2011, 47(21):6006-6008.

10. Cabrita, MA. Focal adhesion kinase inhibitors are potent antiangiogenic agents. Molec. Oncology 2001, 5(6):517-526. 11. Janjetovic, K. Metformin reduces cisplatin-mediated apoptotic death of cancer cells through AMPK-independent activation of Akt. Europ. J. Pharmacol. 2011, 651(1-3):41-50. 12. Shao, J et al. AMP-activated protein kinase (AMPK) activation is involved in chrysin-induced growth inhibition and apoptosis in cultured A549 lung cancer cells. Biochem. BioPhys. Res. Comm. 2012, 423(3):448-453. 13. Ahmed, FE. Toxicology and human health effects following exposure to oxygenated or reformulated gasoline. Tox. Letters 2001, 123(2-3):89-113. 14. Boldingh Debernard, KA. Cell death induced by novel procaspase-3 activators can be reduced by growth factors. Biochem. Biophys. Res. Comm. 2011, 413(2):364-369. 15. Garcia-Martinez, JM. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). Biochem. J. 2009, 421(1):29-42. 16. Strittmatter, F et al. Activation of protein kinase B/Akt by alpha1adrenoceptors in the human prostate. Life Sciences 2012, 90(1112):446-453. 17. Ballou, LM et al. Inhibition of Mammalian Target of Rapamycin Signaling by 2-(Morpholin-1-yl)pyrimido[2,1-a]isoquinolin-4-one. J. Biol. Chem. 2007, 282(33):24463-70.

Kinetic Caspase-3/7 and Annexin Apoptosis Assays Quantitative assays for apoptotic pathway analysis for both drug discovery and basic research

Introduction Apoptosis, the biological process by which cells undergo programmed cell death, is required for normal tissue maintenance and development. However, aberrations in apoptotic signaling networks are implicated in numerous human diseases including neurodegeneration and cancer.1 Apoptotic pathways are initiated by extrinsic factors that result in activation of pro-apoptotic receptors on the cell surface, or intrinsically by many different stimuli such as DNA damage, hypoxia, the absence of growth factors, defective cell cycle control, or other types of cellular stress that result in release of cytochrome C from mitochondria. Stimulation of either the extrinsic or intrinsic apoptotic pathways triggers a signaling cascade that results in the activation of a family of proteins that play a major role in carrying out the apoptotic process called caspases.2 Caspases (cysteinyl aspartate proteinases) cleave substrates following an Asp (D) amino acid residue. Effector targets of caspases include caspase family members themselves, proteins involved in fragmentation of cellular DNA (caspaseactivated DNAses), nuclear lamins, as well as proteins that make up the cell cytoskeleton. Caspase proteins are traditionally separated into two groups, initiator caspases (caspase 2, 8, 9 and 10), and executioner or effector caspases (caspase 3, 6, and 7). As a primary executioner caspase in most systems, the activation of caspase-3 often results in the irreversible commitment of a cell to apoptosis. Therefore, the activation of caspase-3 is considered a reliable marker for cells undergoing apoptosis.

The regulated loss of plasma membrane phosphatidylserine (PS) symmetry is also a classical marker of apoptosis. Dying cells trigger the translocation of the normally inward-facing PS to the cellular surface, allowing for early phagocytic recognition of the dying cell by surrounding phagocytes. Numerous enzymatic, plate-reader and flow-cytometric assays have been designed to measure caspase-3/7 activation or PS externalization. Most caspase-3/7 assays involve luciferase, colorimetric or fluorometric reagent substrates that incorporate a DEVD (Asp-Glu-Val-Asp) peptide motif3 which is recognized by the enzyme. Annexin V is a recombinant protein with a high affinity and selectivity for PS residues, allowing it to be used for the detection of apoptosis. Apoptosis assays using annexin V conjugated to a fluoroprobe have been optimized for detection of PS externalization and are most commonly measured by flowcytometry. In this chapter, we will examine kinetic approaches for measuring apoptosis using IncuCyteÂŽ Caspase-3/7 and Annexin V fluorescent reagents. Unlike plate reader and flow-cytometric endpoint approaches, kinetic live-cell image-based analysis allows for the evaluation of time-dependent effects of treatments or cellspecific responses.

Live-Cell Analysis Handbook — Second Edition

Kinetic Caspase-3/7 and Annexin Apoptosis Assays

Live-cell Apoptosis Assays at a Glance The IncuCyte® Caspsase-3/7 reagent is an inert, non-fluorescent substrate. When added to tissue culture growth medium, the substrate freely crosses the cell membrane where it is cleaved by activated caspase-3/7 resulting in the release of the DNA dye and green fluorescent labeling of DNA.4, 5 IncuCyte® Annexin V reagents are specially formulated, highly-selective cyanine-based fluorescent dyes ideally suited to a simple mix-and-read, real-time quantification of apoptosis in living cells. •• The apoptotic signal relies on either the activation of Caspase-3/7, a primary and irreversible “executioner” pathway in most cell types, or using Annexin V conjugated to a fluoroprobe for detection of PS externalization.

Sample Results •• Multiplexing IncuCyte apoptosis reagents with a nuclearlabeling reagent such as IncuCyte NucLight enables differentiation between inhibition of cell growth and induction of cell death. •• High definition phase contrast images provide an additional qualitative validation of cell death based on morphological characteristics. •• Images are automatically acquired and analyzed to reveal concentration and time-dependent effects on biology.

•• Multiplexing the Caspase-3/7 and Annexin V reagents detect and confirm apoptosis through two different pathways, verifying apoptosis as a mechanism of cell death.

Shortcomings of Traditional Assays

Live-Cell Imaging and Analysis Approaches

• Assays result in a single, user-defined time point measurement of caspase-3/7 activity.

• The assay provides a full kinetic readout of apoptotic signaling over multiple days, eliminating the need for determining a single, optimum, assay endpoint a-priori which can vary considerably for different cell types and for different compound treatment conditions.

• Techniques require multiple wash steps or cell lifting prior to data collection that may result in the loss of dying cells or lead to a loss in PS asymmetry. • Assays are not amenable to long-term measurements due to increasing signal background over time. • Manipulations can result in the loss of cells or critical data in experiments where cells undergo apoptosis at different rates according to treatment conditions.

• Cells can be simultaneously labeled with an IncuCyte apoptosis reagent and NucLight live-cell nuclear labeling reagent to measure apoptotic cell death, cell proliferation and kinetically monitor anti-proliferative effects of compounds. • Addition of IncuCyte apoptosis reagents to normal, healthy cells are nonperturbing to cell growth or morphology and yield little to no intrinsic fluorescent signal. • IC50 and EC50 values can be calculated using kinetic area under the curve values of nuclear counts and caspase-3/7 or annexin V counts, respectively. • 96- and 384-well assay using a homogeneous “mix and read” protocol which can be run over multiple days in full media. No wash or lifting steps required, negating the concern that cells are lost during the experiment or labeling process.

Quantitative measurement of caspase-3/7 kinetic activation MDA-MB-231 cells, a human breast adenocarcinoma derived cell line, were treated with staurosporine (SSP) a well-known inducer of apoptosis. SSP was serially diluted in growth media containing 5 μM IncuCyte Caspase 3/7 reagent in a 96-well plate. Once treated, the cells were immediately placed inside the IncuCyte live-cell analysis system with a 10X objective in a standard cell culture incubator and both phase-contrast and fluorescent images were collected every 2-3 hours.

Alterations in cell morphology were evident within only a few hours of SSP treatment as illustrated in the phase image in Figure 1A. Using fluorescent images, we positively identified cells containing fluorescently stained DNA indicating activation of caspase-3/7, cleavage of the DEVD moiety in the kinetic apoptosis reagent, and fluorescent labeling of cellular DNA (green image in Figure 1A). Using the object counting algorithm, we successfully quantified the number of fluorescent objects , shown in Figure 1A. The object counting criteria were then applied to all images in the experiment at each time point. The data in Figure 1B indicate that caspase-3/7 activation is detectable within a few hours of SSP treatment, with a maximal response triggered in the presence of 333 nM SSP.

Increasing concentrations of SSP also significantly affected cell proliferation. To demonstrate this on the IncuCyte, we completed an end point analysis at the 48 hour time point. Vybrant® DyeCycle™ Green DNA dye was added directly (no wash required) to the wells at a final concentration of 1 μM in 50 μL of PBS. After a 30 minute incubation, the total number of DNA containing objects was enumerated using the object counting algorithm. As expected, our data indicate an inverse correlation between the total number of objects and the apoptotic index as a function of increasing concentrations of SSP (Figure 1C). The data clearly indicate the ability of the IncuCyte system to accurately identify the activation of Caspase-3/7, thus providing insight into the dynamics and timing of the apoptotic signaling pathway, thereby alleviating the need to pick an end-point for analysis prior to running the experiment.

• The assay has high statistical reproducibility and can be used both for singlepoint screening or concentration response profiling. • All data points and temporal data curves can be validated by individual images or time-lapse movies respectively. The kinetic readout of the IncuCyte® system provides both high definition (HD) phase as well as quantitative fluorescent imaging.

Figure 1: Staurosporine (SSP) induced caspase-3/7 activity in human breast adenocarcinoma cells (MDA-MB-231). (A) Representative phase contrast and fluorescent images reveal classical apoptotic cell morphologies and indicate activation of caspase-3/7, respectively. (B) Kinetic measures of the number of caspase-3/7 positive cells is recorded over time and plotted as fluorescent objects, n=3 wells per data point shown (C) At the 48 hour end point, the apoptotic index was calculated by dividing the number of caspase- 3/7 fluorescent objects by the total number of DNA containing objects following staining with Vybrant DyeCycle Green.

Multiplexed, kinetic measurements of proliferation and apoptosis The fluorescent channels on the IncuCyte live-cell analysis system provide a way to kinetically measure caspase-3/7 activation in addition to proliferation (nuclear label) within the same well, eliminating the need for end-point analysis. In the next experiment, HeLa NucLight Red cells were treated with SSP in the presence of 5µM IncuCyte Caspase-3/7 reagent and phasecontrast, red, and green images were collected every 2 hours in the IncuCyte system using a 10x objective (Figure 2). These data illustrate typical results obtained using the IncuCyte Caspase-3/7

reagent multiplexed with IncuCyte NucLight Red cells to measure the kinetic induction of apoptosis and proliferative effects of drug treatment (Figure 2B and C, respectively). Using all of the kinetic data in Figure 2B and 2C, area under the curve (AUC) values were plotted and EC50 (apoptosis) and IC50 (proliferation) values were calculated. This 2-color kinetic assay provides a multiplex way to analyze the apoptotic and anti-proliferative effects of various treatments.

Live-Cell Analysis Handbook — Second Edition A

Kinetic Caspase-3/7 and Annexin Apoptosis Assays

Using images and movies to confirm signaling

One of the major advantages of live-cell analysis is the ability to verify the quantified kinetic data with both phase contrast and fluorescent images. Classical morphological changes associated with apoptosis include: cell shrinkage, membrane blebbing, nuclear condensation, and DNA fragmentation. The time lapse sequence presented in Figure 4 highlights this advantage, illustrating the ability to use phase contrast and fluorescent blended images to temporally correlate the activation of caspase-3/7 and the loss

D A

Figure 2: Pharmacological analysis of caspase-3/7 activation and nuclear counts in HeLa NucLight Red cells treated with SSP. (A) Blended phase-contrast and red/green images taken at 20x show red nuclear signal and activation of caspase-3/7 as well as morphological differences in untreated cells (left) vs. cells treated with 300nM SSP (right). (B) Caspase-3/7 positive objects and (C) nuclear counts were measured over time in response to increasing concentrations of SSP. (D) Area under the curve (AUC) of nuclear counts/mm2 and caspase object counts/mm2 over time were measured and used to calculate IC50 and EC50 values, respectively.

caspase 3 mediated signaling pathways. To demonstrate this using the IncuCyte live-cell analysis system, A549 epithelial carcinoma cells were treated with increasing concentrations of TNF-α in the presence of 5μg/mL CHX. Caspase-3/7 was activated in a TNF-α concentration dependent manner (Figure 3A). The AUC values for caspase-3/7 positive objects over time were then used to calculate the EC50 value of 0.676 nM TNF-α (Figure 3B).

Figure 4: Time-lapse images and movies to detect SSP induced apoptosis in HT-1080 cells. HT-1080 NucLight Red cells were treated with 300nM SSP in the presence of 5μM Caspase-3/7 reagent and imaged in IncuCyte live-cell analysis system every 3 hours (A). Time-lapse images and movies monitor changes in morphology and confirm the activation of the green caspase-3/7 signal in (B) and the loss of the red nuclear signal (C).

Loss of phosphatidylserine asymmetry

Kinetic measurement of extrinsic activation of apoptosis Depending on the cellular context, exposure of cells to tumor necrosis factor alpha (TNF-α) can induce either pro-survival, or cell death pathways. When used in isolation, TNF-α induces NFκB activity and subsequent expression of pro-survival signaling molecules (e.g. FLIP, XIAP, A20). Alternatively, when used in conjunction with cycloheximide (CHX), itself an inhibitor of translation, TNF-α is a potent inducer of apoptosis through

of red nuclei due to cell death with morphological changes in response to treatment with SSP. Using the IncuCyte system, the temporal responses in every well can be supplemented with a “movie” of either a phase contrast, fluorescence or blended timelapse sequence. This ability significantly enhances the confidence in the measured response and any subsequent conclusions drawn from quantitative image analysis.

Addition of the IncuCyte Annexin V reagent to normal healthy cells is non-perturbing to cell growth or morphology and yields little or no intrinsic fluorescent signal. Once cells become apoptotic, plasma membrane PS asymmetry is lost leading to exposure of PS to the extracellular surface and binding of the IncuCyte Annexin V reagent, yielding a bright and photostable fluorescent signal.

B B

Figure 3: Extrinsic activation of caspase-3/7 in A549 lung epithelial cells. (A) Caspase-3/7 activation of A549 lung epithelial cells in response to varying concentrations of TNF-α in the presence of 5μg/mL cycloheximide (CHX). (B) The EC50 value of TNF-α was calculated by using the area under the curve (AUC) of caspase-3/7 objects/mm2 over time. 24

Figure 5: Concentration and time-dependent loss of PS asymmetry. Annexin V reagent was added to Jurkat human T-cell leukemia cells treated with the topoisomerase inhibitor camptothecin. (A) Time-course for the effects of camptothecin on Jurkat cell death (Red Object Confluence (%) presented as the mean ± SEM, n=3 wells). (B) Concentration response curve to camptothecin. Area under the curve (AUC) values have been determined from the time-course shown in panel A (0-36 hours) and are presented as the mean ± SEM, n=3 wells. Average AUC values were used to calculate pIC50 values (camptothecin pIC50 = 8.01). 25

Live-Cell Analysis Handbook â&#x20AC;&#x201D; Second Edition

Kinetic Caspase-3/7 and Annexin Apoptosis Assays

Multiplex Assays to confirm apoptotic pathways

Assessment of immune cell killing of cancer cells using caspase signaling

The IncuCyte system can be used to detect and confirm apoptosis through two different pathways by multiplexing caspase-3/7 and annexin V reagents to verify apoptosis as a mechanism of cell death.

Capsase-3/7 was used to evaluate targeted tumor cell death in a co-culture Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) assay to measure tumor cell apoptosis and for visual validation of tumor-immune cell interactions. To demonstrate the utility of this approach, ADCC cell death was measured via Caspase-3/7 induction in Her2 positive SKOV3 or negative A549 NucLight Red cells (1.6K/well) co-cultured with with PBMCs (8K/well) in the presence of trastuzumab, a clinically used monoclonal antibody for Her-2 positive sold cancers.

Caspase-3/7

Annexin V

Label-free confluence

Proliferation

A concentration-dependent decrease in proliferation (IC50 8.1 ng/mL) and increase in apoptosis (IC50 4.6 ng/mL) was measured in Her2-positive SKOV3 cells. No response was seen in Her2-negative A549 cells (data not shown). These data demonstrate that live cell imaging can be used to discern the full-time course and specificity of immune cell killing, which are traditionally conducted as flow cytometry or biochemical readouts, requiring selection of end points and lack visual confirmation of cellular interactions.

Apoptosis

Figure 6: Detect both caspase-3/7 and annexin V signals in cultures. Automatically determine the time courses of apoptotic cell death and correlate with label free confluence measurements to provide an estimate of the proportion of apoptotic cells within the population (apoptotic index).

Figure 7. Trastuzumab induced ADCC in Her2 positive SKOV3 cells. SK-OV-3 cancer cells were seeded in combination with PBMCs in the presence of trastuzumab to induce ADCC. A concentration-dependent decrease in proliferation (IC50 8.1 ng/mL) and an increase in apoptosis (IC50 4.6 ng/mL) was seen. 26

Live-Cell Analysis Handbook — Second Edition

Conclusions Live-cell imaging and analysis is a powerful tool for the kinetic detection of apoptosis, demonstrating quantitative and reproducible data. This strategy of live-cell imaging and analysis gives the user the ability to monitor morphological changes in parallel with quantification, the combination of which is a powerful and unique tool for detecting pharmacological or genetic manipulations that alter cell health. In addition, NucLight reagents or cell lines, when used in conjunction with IncuCyte apoptosis reagents and the IncuCyte system, provide an additional parameter for measuring cytostatic (anti-proliferative) and apoptotic events. Together, these attributes provide new and unique assays for apoptotic pathway analysis for both drug discovery as well as basic cell biology research.

Key features of the apoptosis assays are: •• The irreversible “executioner” caspase-3/7 pathway or PS externalization can be kinetically monitored allowing for the detection of both short-term and long-term treatment effects. •• Non-perturbing IncuCyte Caspase-3/7 or Annexin V reagents are added as mix-and-read reagents directly to the cultured cells in complete growth media, removing the need for fluid aspiration steps, thus eliminating cell disruption or loss of impaired cells. •• All data points can be validated by individual images or timelapse movies to confirm processing metrics, significantly enhancing the confidence in the measured response. •• Live-cell imaging of apoptosis can be used to discern the full-time course and specificity of co-culture models such as immune cell killing, with visual confirmation of cellular interactions.

References 1.

Cotter TG: Apoptosis and Cancer: The Genesis of a Research Field. Nat Rev Cancer 2009, 9(7):501-507.

Shi Y: Mechanisms of Caspase Activation and Inhibition During Apoptosis. Mol Cell 2002, 9(3):459-470.

Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, GarciaCalvo M, Houtzager VM, Nordstrom PA, Roy S, Vaillancourt JP, Chapman KT, Nicholson DW: A Combinatorial Approach Defines Specificities of Members of the Caspase Family and Granzyme B. Functional Relationships Established for Key Mediators of Apoptosis. J Biol Chem 1997, 272(29):17907-17911. Daya S, Robets M, Isherwood B, Ingleston-Orme A, Caie P, Teobald I, Eagle R, Carragher N: Integrating an Automated in Vitro Combination Screening Platform with Live-Cell and Endpoint Phenotypic Assays to Support the Testing of Drug Combinations. SBS 16th Annual Conference and Exhibition 2010.

Cen H, Mao F, Aronchik I, Fuentes RJ, Firestone GL: Devd-Nucview488: A Novel Class of Enzyme Substrates for Real-Time Detection of Caspase-3 Activity in Live Cells. FASEB J 2008, 22(7):2243-2252.

Janicke RU, Sprengart ML, Wati MR, Porter AG: Caspase-3 Is Required for DNA Fragmentation and Morphological Changes Associated with Apoptosis. J Biol Chem 1998, 273(16):9357-9360.

Zhang JH, Chung TD, Oldenburg KR: A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 1999, 4(2):67-73.

Kinetic Cytotoxicity Assays Real-time, live-cell assay to quantify and visualize cytotoxic events

Introduction

IncuCyte® Cytotoxicity Assay at a Glance

The cellular response to cytotoxic exposure is controlled by complex biochemical pathways, such as necrosis or apoptosis, which results in cell death. In apoptosis, morphological changes include pseudopodia retraction, reduction of cellular volume (pyknosis), nuclear fragmentation (karyorrhexis) and eventually loss of plasma membrane integrity.1 Morphological changes that characterize necrosis include cytoplasmic swelling and early rupture of plasma membrane.2 Compounds that have cytotoxic effects often compromise cell membrane integrity regardless of the pathway.

In order to measure cell membrane integrity over time while simultaneously monitoring associated morphological changes, IncuCyte® CytoTox Red and Green reagents do not affect cell growth or morphology and have been optimized for the kinetic detection of cytotoxicity in a no-wash, mix-and-read assay. CytoTox reagents are cell impermeant cyanine dimer nucleic acid stains that bind to dsDNA,5 and when added to the culture medium, these reagents fluorescently stain the nuclear DNA of cells that have lost plasma membrane integrity.

Assays designed to measure cytotoxicity in vitro are used to predict tissue-specific toxicity or to identify and classify leads for anti-cancer therapies. Multiplexed, high-throughput screening (HTS) in vitro cytotoxicity assays measuring a variety of different readouts are being employed to assess the cytotoxicity of compounds in early drug development. 3 Commonly used cytotoxicity assays evaluate a range of end-point parameters, such as the release of lactate dehydrogenase (LDH) and glutathione (GSH) following membrane rupture, generation of reactive oxygen species (ROS), cell proliferation, and disruption of mitochondrial trans-membrane potential. Critical factors contributing to the predictive nature of these assays include compound concentration, and more importantly, the time allowed for the compound to elicit an effect.4 Although these multiplexed assays are able to simultaneously measure multiple indicators of in vitro cytotoxicity, they typically assess a single time point and are unable to assess the biological activity over time.

This cytotoxicity assay can be combined with IncuCyte® NucLight cell labeling reagents that incorporate a red or green nuclear label allowing for simultaneous measurement of proliferation and cytotoxicity in a single well. Non-perturbing NucLight reagents provide a means to kinetically quantify cell proliferation over time. The reagents allow for the expression of a nuclear-restricted GFP (green fluorescent protein) or mKate2 (red fluorescent protein) in mammalian cells without altering cell function and with minimal toxicity. NucLight lentivirus reagents allow for the creation of stable cell populations or clones. Phase-contrast images can be used to qualitatively monitor associated morphological changes in the same cells over the same time course.

In this chapter, we will examine kinetic approaches for measuring cytotoxicity using reagents that fluorescently stain the nuclear DNA of cells that have lost plasma membrane integrity. Unlike traditional endpoint approaches, kinetic live-cell imaging allows for the analysis of time-dependent variation in treatment response as well as the ability to differentiate between cytostatic and cytotoxic effects.

Live-Cell Analysis Handbook — Second Edition

Kinetic Cytotoxicity Assays

Shortcomings of Traditional Assays

Live-Cell Imaging and Analysis Approaches

• Assays result in a single, user-defined time point measurement.

• The assay provides a full kinetic readout of cytotoxicity over multiple days, eliminating the need for determining a single, optimum, assay endpoint a-priori which can vary considerably for different cell types and for different compound treatment conditions.

• Lack of ability to assess biological activity over time limits predictive nature. • Manipulations can result in the loss of cells or critical data in experiments where cells undergo cell death at different rates according to treatment conditions.

In contrast, no statistical induction of cytotoxicity was observed when either cell type was treated with any of the tested concentrations of CHX (Figure 1A, B). However, a clear concentration-dependent inhibition of cell proliferation was observed as measured by the NucLight Red fluorescent signal

• Addition of IncuCyte CytoTox reagents to normal, healthy cells are nonperturbing to cell growth and morphology.

(Figure 2). Moreover, end point normalization, which corrects for differences in proliferation within treatment groups, revealed a concentration-dependent cytotoxic index for both SSP and CMP in MDA-MB-231 and HT-1080 cells, whereas no cytotoxic responses was induced by treatment with CHX (Figure 3).

Membrane Permeability

• Cells can be simultaneously labeled with an IncuCyte CytoTox reagent and NucLight nuclear labeling reagent to measure cytotoxicity and cell proliferation.

Proliferation

• 96- and 384-well assay format follows a homogeneous “mix and read” protocol which can be run over multiple days in full media. No wash or lifting steps required, negating the concern that cells are lost during the experiment or labeling process. • All data points and temporal data curves can be validated by individual images or timelapse movies respectively. The kinetic readout of the IncuCyte® system provides both high definition (HD) phase as well as quantitative fluorescent imaging.

Sample Results Quantitative measurement of cytotoxicity using IncuCyte CytoTox reagent Staurosporine (SSP) and camptothecin (CMP) are compounds that are known to cause cell death due to cytotoxicity. SSP is a high affinity, non-selective, ATP-competitive kinase inhibitor and is classically used as a research tool to induce caspase-3 mediated apoptosis.6, 7 CMP causes cell death by inhibition of the DNA enzyme, topoisomerase I (topo I), resulting in double strand breaks during S-phase and triggering the apoptotic program.8 These two compounds were used to illustrate the ability of the cell impermeant DNA dye based cytotoxicity assay to measure cell death over-time using two different cell lines, HT 1080 (human tumor derived fibrosarcoma) and MDA-MB-231 (human tumor derived breast adenocarcinoma. In addition, we also measured the response of these same two cell types to the protein synthesis

MDA-MB-231

inhibitor, cycloheximide (CHX), a cytostatic compound which was predicted to inhibit cell proliferation while not affecting cell viability (Figure 1). A 7-point concentration curve of each compound clearly illustrated that in both cell types, SSP and CMP induced a concentration-dependent cytotoxic response. Specifically, we observed a statistical induction of cytotoxicity in MDA-MB-231 cells at 16 and 26 hours for SSP and CMP treatments, respectively (Figure 1A). In identically treated HT 1080 cells, there was a more rapid induction of the cytotoxic responses correlating to 12 and 22 hours for SSP and CMP treatments, respectively, which illustrates a slight cell type dependent difference (Figure 1B).

HT 1080

Figure 1. Discrimination of cytotoxic and cytostatic compounds. 96-well microplate graph showing the kinetic measurement of cell death as determined by CytoTox Green staining in response to several concentrations of SSP, CMP, and CHX in MDA-MB-231 cells (A) and HT 1080 cells (B). 30

Figure 2. Cytostatic effect of cycloheximide (CHX). NucLight Red HT-1080 cells were treated with several concentrations of cycloheximide in the presence of CytoTox Green. Graphs illustrate no induction of cytotoxicity as measured by green fluorescence staining of DNA (A) however, inhibition of cell proliferation (B) as measured by fluorescent nuclear counts is observed. Cell morphology did not significantly differ from untreated cells as illustrated in Figure 5. Each data point represents the mean ± SE in N=3 wells.

MDA-MB-231

HT 1080

Figure 3. End point normalization of cytotoxic and cytostatic compounds. At the 72-hour end point, Triton X-100 at a final concentration of 0.0625% was added to allow nuclear dsDNA staining by CytoTox Green of all cells present/well. The cytotoxic index was calculated by dividing the number of CytoTox Green fluorescent objects by the total number of DNA containing objects (fluorescent objects counted post Triton X-100 treatment). 31

Live-Cell Analysis Handbook — Second Edition SSP

CMP

Kinetic Cytotoxicity Assays CHX

Control

B Figure 4. Morphological images. Phase-contrast and fluorescent images of NucLight Red HT-1080 cells 24hrs post-treatment, showing morphological changes in response to SSP, CMP and CHX.

The IncuCyte® system acquired phase-contrast and fluorescent images of HT-1080 cells, under all three compound treatments, confirmed fluorescent object count data as illustrated in Figure 4 (similar results were observed with MDA-MB-231 cells). These data show the potential of this kinetic and morphological approach to the screening, prioritization, and classification of compounds in drug discovery. In order to evaluate the accuracy and reproducibility a series of experiments using MDA-MB-231 and HT-1080 cell lines and two known cytotoxic compounds (SSP and CMP) were used.

To assess intra-assay reproducibility, each well of a 96-well plate was seeded with 5,000 HT-1080 or MDA-MB-231 cells. Each row of cells was treated with 2-fold decreasing concentrations of CMP (2000 nM to 62.5 nM; n=12 wells per condition) in the presence of CytoTox Green and kinetic measurements of fluorescent objects were analyzed. As illustrated in the microplate graph view in Figure 5A, we obtained a highly reproducible kinetic measure of cell viability. In addition, using end point data, we were able to demonstrate an inverse relationship between total DNA object count and cytotoxic index (Figure 5B, C) in both cell lines analyzed.

MDA-MB-231

Figure 6. Statistical analysis of intra-assay reproducibility. Calculated cytotoxic index of HT-1080 (A) and MD-MB-231 (B) cells to decreasing concentrations of CMP. (C) EC50 values determined from HT-1080 plate described in Figure 5A.

Assay Z’ factors of 0.82 (HT 1080) and 0.64 (MD-MB-231) were calculated, indicating that this assay platform is amenable to screening protocols (Figure 6A, B). In addition, using the end point cytotoxic index from the HT-1080 data, we were able to calculate remarkably consistent pEC50 values from each column of the microplate with a total geometric mean of 198 nM (Figure 6C). In order to determine inter-assay reproducibility and accuracy, HT-1080 cells were seeded in two 96-well plates at a density

A A

Inter-Assay Reproducibilty

CytoTox Green fluorescent objects/mm2 (HT 1080)

Figure 5. Intra-assay reproducibility of HT-1080 and MDA-MB-231 cells in response to CMP. 96-well microplate graph showing reproducibility of concentration response to CMP (A). After the 48-hour end point, the cytotoxic index was calculated by dividing the number of CytoTox Green fluorescent objects by the total number of DNA containing objects (fluorescent objects counted post Triton X-100 treatment) (B and C).

of 5,000 cells/well. Random wells of each plate were spiked with independently prepared stocks of CMP at 1000, 500, 200 and 50 nM (n=6 per concentration), as illustrated in Figure 7A. The data from both plates were then plotted on different axes and analyzed using linear regression (Figure 7B). The resulting R2 value of 0.9588 demonstrates a strong correlation between identically treated wells on separate plates. Additionally, the Z’ factors of 0.72 and 0.62 are again indicative of a high quality assay (Figure 7C).

HT 1080

Figure 7. Inter-assay reproducibility of HT-1080 response to CMP. At the 72-hour end point, Triton X-100 at a final concentration of 0.0625% was added to allow nuclear dsDNA staining by CytoTox Green of all cells present/well. The cytotoxic index was calculated by dividing the number of CytoTox Green fluorescent objects by the total number of DNA containing objects (fluorescent objects counted post Triton X-100 treatment). (A) A 96-well microplate graph showing reproducibility of singe-well responses to various concentrations of CMP on HT-1080 cells. (B) Replicate plates of HT-1080 were spiked with identical concentrations of CMP and results were graphed to show correlation. (C) Statistical measurements from the same plates showing Z’ factors exceeding 0.60. 33

Live-Cell Analysis Handbook — Second Edition

Kinetic Cytotoxicity Assays

Differentiating cytotoxic and cytostatic treatments A multiplexed assay capable of measuring cytotoxicity in addition to cell proliferation was created. HT-1080 NucLight Red cells were seeded at 5,000 cells/well and treated with serially diluted concentrations of SSP, CMP or CHX in the presence of 0.1 μM CytoTox Green (all data not shown). The IncuCyte system was used to mask the green fluorescent nuclear signal to quantitate cell death as well as the red fluorescent nuclear signal to monitor cell proliferation kinetic dose response curves for both CytoTox Green positive events as well as nuclear counts of NucLight Red HT-1080 cells were exported to GraphPad Prism. Statistical analysis of the area under the curve (AUC) was calculated for time points within the kinetic curves. Replicate AUC values, at peak response, were used to calculate EC50 and

IC50 values. Figure 8 shows the inverse relationship between cell proliferation (nuclear count) and membrane permeability (CytoTox Green positive objects) over time in the presence of SSP. The AUCs were then used to statistically determine at which concentration SSP exhibited purely cytostatic effects. The concentration at which the AUC of CytoTox Green objects/mm2 over time was different from the control group was termed cytotoxic and/or cytostatic. Concentrations where the AUC of CytoTox Green objects/mm2 over time was not different from that of the control, but the AUC of nuclear objects/mm2 over time was significantly different from the control was termed purely cytostatic. These data show the potential of this kinetic and multi-parametric approach to the classification of compounds in drug discovery.

References 1.

Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G: Cell death assays for drug discovery. Nat Rev Drug Discov 2011, 10(3):221-237.

Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny MV, El- Deiry WS, Golstein P, Green DR et al: Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 2009, 16(1):3-11.

Abraham VC, Towne DL, Waring JF, Warrior U, Burns DJ: Application of a high-content multiparameter cytotoxicity assay to prioritize compounds based on toxicity potential in humans. J Biomol Screen 2008, 13(6):527-537.

Abassi YA, Xi B, Zhang W, Ye P, Kirstein SL, Gaylord MR, Feinstein SC, Wang X, Xu X: Kinetic cell-based morphological screening: prediction of mechanism of compound action and off-target effects. Chem Biol 2009, 16(7):712-723.

Becker B, Clapper J, Harkins KR, Olson JA: In situ screening assay for cell viability using a dimeric cyanine nucleic acid stain. Anal Biochem 1994, 221(1):78-84.

Chae HJ, Kang JS, Byun JO, Han KS, Kim DU, Oh SM, Kim HM, Chae SW, Kim HR: Molecular mechanism of staurosporine-induced apoptosis in osteoblasts. Pharmacol Res 2000, 42(4):373-381.

Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT, Chan KW, Ciceri P, Davis MI, Edeen PT et al: A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 2008, 26(1):127-132.

Figure 8. Inter-assay reproducibility of HT1080 response to CMP. HT-1080 cells were treated with varying concentrations SSP. Dual fluorescent images were used to calculate the area under the curve (AUC) of cell death (CytoTox Green Object Cnt/mm2) and cell proliferation (Nuclear Cnt/mm2) over time. Average AUC values were then used to calculate EC50 and IC50 values, respectively. Dunnett’s Multiple Comparison Test was used to compare the differences between AUCs at each concentration.

Conclusions Using the IncuCyte system in conjunction with the IncuCyte CytoTox Green or Red reagents as a live-cell, kinetic assay for the measurement of cytotoxicity has demonstrated quantitative and reproducible detection of cell permeability, a hallmark of cell death. This strategy also gives the user the ability to monitor morphological changes in parallel with quantification, the combination of which is a powerful and unique tool for detecting pharmacological or genetic manipulations that alter cell viability. In addition, NucLight reagents or cells lines, when used with IncuCyte CytoTox Green or Red reagents and the IncuCyte live-cell analysis system, provide an additional parameter for measuring cytostatic (anti-proliferative) and cytotoxic events.

Key features of the cytotoxicity assay are: •• Non-perturbing cytotoxicity reagents are added directly to the cultured cells, removing the need for fluid aspiration steps, thus eliminating cell disruption or loss of impaired cells. •• Data generated using live-cell multi-parametric analysis of cytotoxicity and proliferation delivers more reliable data and allows for differentiation between cytosatic and cytotoxic effects of treatments as well as detection of both short-term and long-term alterations. •• All data points can be validated by individual images or timelapse movies to confirm processing metrics, significantly enhancing the confidence in the measured response.

Live-Cell Analysis Handbook â&#x20AC;&#x201D; Second Edition

Kinetic Cell Migration and Invasion Assays Real-time automated measurements of cell motility

Cell migration and invasion play a role in many normal and pathological processes including immune responses, embryonic development, angiogenesis, regeneration, tumor metastasis, and wound healing.1 A wide variety of assays can be used to monitor and analyze cell migration and invasion in vitro, from very simple assays to very complex. The scratch assay permits velocity measurements of wound closure and is dependent upon a cell density gradient, whereas methods to study chemical factors that regulate migration and invasion of cells typically employ modifications of Boyden chamber assays. Scratch assays are typically performed by manually creating scratches in multiple well plates (e.g., 6-, 12-, 24-or 48-well) and then acquiring images using a microscope at defined time-points. This approach leads to variation in scratch uniformity, as well as disrupting the assay environmental control during image acquisition. Current chemotaxis assay approaches have inherent drawbacks such as non-linear gradients, inability to observe cell morphology and validate cell movement, are technically challenging and have cumbersome quantification steps. In order to drive development of novel therapeutics, a deeper understanding of these processes is required to gain valuable insights. IncuCyte® live-cell migration and invasion assays allow continuous visualization and analysis of cell motility and kinetics. This

Migration Cell Density Dependent (no gradient)

approach addresses the inherent shortcomings of traditional assays that deliver a user-defined time points or a single end-point measurement and don’t allow visualization of cells as they move in response to their environment. The IncuCyte® Scratch Wound Assay is a method that utilizes the IncuCyte® WoundMaker tool to create precise scratches within a cell monolayer, eliminating assay variability that is inherent in “home-brew” methods.2 The IncuCyte® Chemotaxis Assay is a kinetic and visual chemotactic driven method, that alleviates hurdles such as laborious end point measurements, high cell usage, and non-linear gradient that is common in traditional transmembrane assays. These image-based detection methods offer simple and reliable assays which deliver new biological insights into cell migration and invasion.

Live-cell imaging and analysis approaches The IncuCyte cell migration and invasion assays enable the detection and analysis of live-cell movement in 96-well formats. IncuCyte Scratch Wound and ClearView Chemotaxis assays allow you to kinetically monitor and analyze migration and invasion with or without a chemotactic gradient, and associated images allow for the ability to observe cell morphology.

Scratch Wound Assays

Can my cells move across a surface in response to cell density changes?

Can my cells break down a matrix and invade in response to cell density changes?

No matrix Chemical Gradient

Invasion

Matrix (ECM) Can my cells move across a surface in response to a chemical gradient?

Chemotaxis Assays

Can my cells break down a matrix and invade in response to a chemical gradient?

Live-Cell Analysis Handbook — Second Edition

Kinetic Cell Migration and Invasion Assays

How Live-Cell Migration and Invasion Assays Work IncuCyte® Scratch Wound Assay

IncuCyte® Chemotaxis Assay

The IncuCyte Scratch Wound Assay is a 96-well, high throughput wound assay that measures cell density-dependent migration and invasion. Utilizing the IncuCyte® WoundMaker, 96 precise, uniform, cell-free zones are created in cell monolayers and wound closure is visualized and analyzed in real-time with the IncuCyte® live-cell analysis system and software.

The IncuCyte Chemotaxis Assay enables real-time visualization and quantification of cell migration and invasion in response to a chemotactic gradient. Using an optically clear membrane insert that contains optimally spaced 8-micron pores, cell migration and invasion are automatically imaged and analyzed as the cells move through the pores, generating a chemotactic signal.

References 1. 2.

Cell Migration: integrating signals from front to back. Ridley, A.J., Schwartz, M.A., Burridge, K., Firtel, R.A., Ginsberg, M.H., Borisy, G., Parsons. J.T. and Horwitz, A.R. Science 302: 1704. Cell Migration and Invasion Assays as Tools for Drug Discovery. Hulkower, K. I and Herber, R. L. Pharmaceutics 2011 3:107

Recent IncuCyte® Publications

Chemotaxis assay

Taylor, et.al., used the IncuCyte® chemotaxis assay to study netrin-1, a cellular guidance signal. The authors demonstrated that netrin-1 inhibits human monocyte and murine macrophage migration towards C5a, a complement peptide with potent chemotactic and anaphylatoxic properties that upregulate endothelial adhesion molecules, increase vascular permeability, and localize leukocytes and inflammatory molecules at sites of infection. Netrin-1 reduces monocyte and macrophage chemotaxis towards the complement component C5a. Taylor, L., Brodermann, M. H.,McCaffary, D., Iqbal, A. J., Greaves, D. R. PLoS ONE, 2016 Aug 10; 11(8).

A study by Pasqualon, et. al., demonstrated that syndecan-1, a surface-expressed proteoglycan on epithelial tumor cells, promotes macrophage migration inhibitory factor (MIF) binding and MIF-mediated cell migration. Invasion studies showed that MIF induced the chemotactic migration of A549 tumor cells, wound closure and invasion into Matrigel without affecting cell proliferation. These MIFinduced responses were abrogated by silencing of syndecan-1. The authors conclude that this may represent a relevant mechanism through which MIF enhances tumor cell motility and metastasis. Cell surface syndecan-1 contributes to binding and function of macrophage migration inhibitory factor (MIF) on epithelial tumor cells. Pasqualon, T., Lue, H., Groening, S., Pruessmeyer, J., Jahr, H., Denecke, B., Bernhagen, J., Ludwig,A. BBA-Mol Cell Res. 2016 Apr; 1864 (4). Orphan G protein-coupled receptor GPRC5A modulates integrin β1-mediated epithelial cell adhesion. Bulanova, D; Akimov, Y; Rokka, A; Laajala, T; Aittokallio, T; Kouvonen, P; Pellinen, T; Kuznetsov, S. Cell Adhesion & Migration. 2016 Oct. Potent EMT and CSC phenotypes are induced by oncostatin-M in pancreatic cancer. Smigiel, J; Parameswaran, N; Jackson, MMol Can Res. 2017 Jan.

Scratch wound assay

Drebert, et. al., used the IncuCyte scratch wound assay to assess HUVEC migration in response to conditioned medium from solvent- (CMS) and dexamethasone (Dex)-treated (CMD) colon cancer-derived myofibroblasts. In the HUVEC scratch assay CMS-induced acceleration of wound healing was blunted by CMD treatment. Colon cancer-derived myofibroblasts increase endothelial cell migration by glucocorticoid-sensitive secretion of a pro-migratory factor. Drebert, Z., MacAskill, M., Doughty-Shenton, D., De Bosscher, K., Bracke, M., Hadoke, P. W., Beck, I. M. Vascul. Pharmacol., Vascul Pharmacol. 2016 Oct 4. pii: S15371891(16)30137-9. doi: 10.1016/j.vph.2016.10.004. [Epub ahead of print], 2017.

Härmä, et. al., cultured prostate cancer cells on ImageLock plates from Essen BioScience until fully confluent and scratched with the WoundMaker instrument. Wound closure was monitored and quantified with the IncuCyte® system. Study results suggest that some betulin-derivatives may specifically target cell motility and invasion by affecting the organization of filamentous actin fiber network at low nanomolar concentrations. Optimization of invasion-specific effects of betulin derivatives on prostate cancer cells through lead development. Härmä, V., Haavikko, R., Virtanen, J., Ahonen, I., Schukov, H. P., Alakurtti, S., Purev, E., Rischer, H., Yli-Kauhaluoma, J., Moreira, V. M., Nees, M., Oksman-Caldentey, K. M. PLoS ONE 05/2015 10(5):e0126111.

Moody, et. al. conducted scratch wound assays using the IncuCyte platform to assess the functional consequence of growth arrest specific 6 (Gas6) neutralization in lung cancer cells. Gas6 is a vitamin-K dependent, 75 kDa growth factor-like protein involved in the regulation of a wide array of cellular activities, including adhesion, migration, mitogenesis, aggregation, stress-response, differentiation, immune activation, efferocytosis and apoptosis. A Gas6 neutralizing antibody was also tested for its ability to inhibit Gas6-induced proliferation using the same platform. Antibody-mediated neutralization of autocrine Gas6 inhibits the growth of pancreatic ductal adenocarcinoma tumors in vivo. Moody, G., Belmontes, B., Masterman, S., Wang, W., King, C., Murawsky, C., Tsurda, T., Liu, S., Radinsky, R., Beltran, P. Int J of Cancer. 2016 139:1340.

This review article explores how advances in time-resolved imaging contributes to the characterization of distinct modes of invasion and molecular mechanisms. The author highlights the latest advances in kinetic imaging instrumentation applicable to in vitro and in vivo models of tumor invasion. Profiling distinct mechanisms of tumor invasion for drug discovery: imaging adhesion, signalling and matrix turnover. Carragher, N. Clinical & Exp Metastasis. 2009 April 26(4).

Live-Cell Analysis Handbook — Second Edition

Kinetic Scratch Wound Assays

Shortcomings of Traditional Assays

Live-Cell Imaging and Analysis Approaches • Real-time, direct visualization and automated analysis of migration and invasion.

Real-time assays to assess migration and invasion potential

• Assays result in a single, user-defined time point measurement or requires manual intervention to generate multiple time points. • Lack of precision and reproducibility in creating the wound and capturing data.

• 96-well ImageLock plates enable precise, scan to scan repeat imaging of the same field of view. • Reproducible kinetic data supported by images and time-lapse movies. • Spatio-temporal, label-free format provides data on both the rate and the extent of migration and invasion for a given set of experimental variables.

Introduction

IncuCyte® Scratch Wound Assay at a Glance

Cell migration is a multistep process that is a fundamental component of many biological and pathological processes such as embryonic development, tissue re-organization, angiogenesis, immune cell trafficking, chronic inflammation and tumor metastasis. Cell migration is initiated by a stimulus that activates a set of signaling pathways leading to cellular polarization and a rapid reorganization of actin filaments and microtubules. Cells advance by protruding their membrane at their leading cell border, which is followed by dynamic substrate adhesion via integrin adherence to the substrate. Membrane retraction at the lagging cell edge finishes the cycle, which is then repeated in rapid succession. The summation of this process results in cell migration.

The IncuCyte® scratch wound migration and invasion assay can be used to quantitatively assess both cellular migration and cell invasion potential in the presence of experimental agents. The 96-well IncuCyte® WoundMaker creates cell-free zones in cell monolayers cultured on IncuCyte® ImageLock 96-well plates with the use of precision engineered pins, without disrupting the underlying biomatrix or damaging the tissue culture treated plastic. After wounding, the scratch wound assay is initiated by simply adding media to the cells for the migration assay or by overlaying the cells with an optimized concentration of a biomatrix material for the invasion assay. IncuCyte® scratch wound analysis software enables real time, automated measurement of label-free or dual fluorescence of cell migration and invasion.

In this chapter, we will review real-time visualization and analysis of cell movement that is driven by changes in cell density. Unlike traditional assays, which require repeatedly removing cells from an incubator to perform manual image acquisition and have variability in scratch width and uniformity, this live-cell assay approach provides continuous analysis and eliminates assay variability with the use of the WoundMaker tool to create precise scratches in the cell monolayer.

•• Visualize cell migration and invasion, and assess morphological changes in real time with the scratch wound method.

• Explore time-dependent pharmacology, in order to enhance assay sensitivity. • Measure migration without fixing or staining steps.

Sample Results Assessing wound closure rates and morphology The differences in wound closure rates and cell morphology between migrating and invading HT-1080 cells are shown in Figure 1. Migrating HT-1080 cells (odd numbered columns) closed the wound region at a significantly faster rate with complete wound closure detected by 10-12 hours post wounding. In contrast, HT1080 cells invading 8 mg/ml Matrigel® reached 80% wound closure within 48 hours as measured by the Relative Wound Density metric. Morphological differences were also noted. Migrating cells

maintained a fibroblastic morphology, had rounded lamellipodia, and advanced as a uniform population of cells. Invading HT-1080 cells adopted a mesenchymal phenotype displaying extended cell bodies and, in some cases, “spike-like” lamellipodia as the cells advanced into the Matrigel matrix in an irregular manner. The clear morphological differences between migrating and invading HT-1080 cells can be used to select the optimum condition for the invasion assay.

C Migration

•• Assays are flexible, quantitative and easily reproducible with the IncuCyte WoundMaker tool. •• Monitor and quantify cell movements across a substrate (migration) or through a gel matrix (invasion) in the same plate, up to six 96-well plates at once.

Invasion (8mg/mL Matrigel)

•• Validated with a wide range of adherent primary, immortalized and tumor cell types.

Figure 1. Measurement of the reproducibility the migration and invasion assay in the same microplate. HT-1080 cells were plated at 2 x 104 cells per well on 100 μg/ml Matrigel coated ImageLock plates. The cells in odd numbered columns had only media added after using the 96-well WoundMaker representing cell migration. The cells in even numbered columns were overlaid with 8 mg/mL Matrigel representing invasion. (A) Temporal progression of wound closure in each well with time using RWD as the metric to measure migration or invasion. (B) Time course of means of each condition. The respective coefficients of variation for each assay were averaged. (C) Representative images of HT-1080 cells migrating on Matrigel (left) and invading through 8mg/mL.

Differentiating between invasive and non-invasive cells Both MDA-MB-231 and HT-1080 cells are highly invasive cell types. In contrast, MCF-7 cells are relatively non- invasive. All three cell types were tested in invasion and migration assays as indicated in Figure 2. As previously shown, HT-1080 cells migrated on collagen 1 coated plates and invaded collagen 1, with the rate of invasion slowing as the concentration of collagen 1 matrix was increased from 1-3 mg/ml. MDA-MB-231 cells also migrated on collagen 1-coated plates, but invaded collagen more slowly than 40

HT-1080 cells. Interestingly, the rate of invasion of MDA-MB-231 cells was similar at all concentrations of collagen 1 tested. MCF-7 cells, like the other two cells types, migrated on collagen 1-coated plates, but in contrast to the other two cells types, MCF-7 cells did not have the capacity to invade collagen 1 at the concentrations tested.

Live-Cell Analysis Handbook — Second Edition

Kinetic Scratch Wound Assays

Figure 2. 96-well microplate graph of three cell types in the cell migration and invasion assay. All wells were coated with 300 μg/mL collagen 1. HT-1080 (2 x 104 cells per well) are in column 1-4, MDA-MB-231 (2.5 x 104 cells per well) are in column 5-8 and MCF-7 (5 x 104 cells per well) are in column 9-12. Rows A and B show the cell migration data for the three cell types. The same cells in the invasion assay are shown in rows C-D (1 mg/ml collagen 1), rows D-E (2 mg/ml collagen 1) and rows G-H (3 mg/ ml collagen 1). The plate map graph shows the progression of each well with time using the RWD metric to measure migration or invasion.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade collagen 1 and other basement membrane materials and are expressed at increased levels by many highly metastatic tumor derived cells. GM6001 is a broad MMP inhibitor that has been shown to inhibit the invasion of HT-1080 cells into collagen 1. 3 The 96-well Migration and Invasion Assay was configured in order to test the effect of this drug on invasion and migration on the same microplate. As shown in Figure 4A, GM6001 had no effect on the migration of HT-1080 cells. In contrast, as shown in Figure 4B, GM6001 inhibited invasion into collagen 1 in a concentration-dependent manner. Interestingly, the addition of a protease cocktail containing E-64 (25 μM) pepstatin A (100 μM), leupeptin (2 μM) and aportinin (2 μM) had no measurable effect on cell migration or cell invasion (data not shown).

Pharmacology applications Blebbistatin is a pharmacological agent that is known to inhibit myosin by binding to the ATPase intermediate with ADP and phosphate bound at the active site, slowing the release of phosphate and inhibits locomotion of cells.1 Previous studies have suggested that blebbistatin may be more effective at inhibiting cell invasion as compared to cell migration.2 With the 96-well format, it is easy to set up an experiment to measure migration and invasion concurrently within the same microplate. Using this approach, the effect of blebbistatin on migration and invasion using HT-1080 cells was studied. The plate map in Figure 3A demonstrates a convenient way to set up this experiment. Three columns of cells were used for migration, and three were used for invasion. A 7-point concentration curve of blebbistatin was carried

out as depicted in rows A-G. Row H was used a solvent control. The microplate graph in Figure 3B demonstrates the reproducibility of the assay and shows the effect of each concentration of drug in both assay formats. By inspection, it appeared that blebbistatin had a larger effect on invasion compared to migration. Plotting the data as the average of the treatment group for migration and invasion made it clear that blebbistatin had a much larger effect on invasion (Figure 3 C and D, respectively). Figure 3E shows the concentration response analysis at the 24-hour time point for both assays. From these data, the calculated IC50 of blebbistatin for migration and invasion is 92 and 5.2 μM, respectively. The Z’ for this data set is 0.77.

C A

Figure 4. Effect of MMP inhibitor. HT-1080 cells were plated at 2 x 104 cells per well on 300 μg/mL collagen 1 coated plates. The GM6001 concentration response was carried out as described for blebbistatin experiment described in Figure 3. Panel A and B show the means of each treatment group for migration and invasion, respectively. Panel C shows the effect of GM6001 on cell migration and invasion in a bar chart format at the 32-hour time point.

Figure 3. Effect of blebbistatin on the migration and invasion of HT-1080 cells. HT-1080 cells were plated at 2 x 104 cells per well on 300 μg/mL collagen 1 coated plates. Panel A shows the plate map for the experiment. The cells in columns 1-3 were overlaid with 3 mg/mL collagen 1 containing blebbistatin or solvent control. The cells in columns 4-6 were given complete growth media with blebbistatin or the solvent control. Panel B shows the microplate graph of the experiment. Panel C and D show the means of each treatment group for migration and invasion, respectively. Panel E shows the concentration response analysis of blebbistatin on cell migration and invasion. The IC50 calculation for each assay is included next to the concentration response curve. 42

Live-Cell Analysis Handbook — Second Edition

Two-color fluorescent scratch wound experiments In addition to phase contrast images, two-color images can be collected using IncuCyte® fluorescent labeling reagents. This allows study of the interactions between several cell types in a mixed culture, and how each affects migration, invasion, and proliferation of the other, all within one well of a 96-well plate. As shown in Figure 5, the non-invasive MCF- 7 cells were labeled

with IncuCyte® NucLight Green Lentivirus Reagent, and the HT-1080 cells labeled with IncuCyte® NucLight Red Lentivirus Reagent were mixed in co-culture, and plated for an invasion assay through 8 mg/ml Matrigel®. Imaging in phase, red, and green channels revealed the HT- 1080 cells efficiently invaded the Matrigel® matrix whereas MCF-7 remained non-invasive.

Figure 5. Two color fluorescent scratch wound. The ability to image cells in both wavelengths in addition to phase contrast allows users the ability to explore cell-cell interactions as it pertains to cell migration and invasion. In this example, HT-1080 NucLight Red cells were plated with MCF-7 NucLight Green cells and invasion through 8 mg/ml Matrigel® was monitored over time (Image shows the 24 hour time point).

Conclusions IncuCyte scratch wound migration and invasion assays are flexible, quantitative, and reproducible. The unique tip design of the 96-well IncuCyte WoundMaker is a critical part of these assays, as it creates a cell-free zone from a confluent monolayer of cells making the biology consistent and reproducible. Both assays utilize IncuCyte’s innovative high definition optics and integrative analysis algorithms to make quantitative measurements without the need for labeling cells. Having high-quality images at every time point gives an investigator access to both the kinetics and morphological changes occurring in migration and invasion experiments, enabling additional insight intothe effects test agents have on cells undergoing these processes. Key features and benefits include:

•• The spatio-temporal, label-free format of both the Migration and Invasion Assays allows investigators to follow both the rate and the extent of migration and invasion for a given set of experimental variables. This feature can be used to explore time-dependent pharmacology, in order to enhance assay sensitivity.

•• Investigators can measure migration and invasion on the same microplate. This provides the best opportunity for determining the specificity of drugs and the utility of potential drug targets.

•• HD images are acquired at every time point and can be automatically assembled into time-lapse movies for convenient visualization of morphology and wound closure.

•• After the experiment is initiated, phase contrast and/ or fluorescence images are collected and processed automatically, yielding un-biased results. •• Precise wounding ensures that results are quantitative and reproducible. •• IncuCyte high definition optics eliminates the need to label the cells.

References 1.

Kovacs, M., Toth, J,, Hetnyi, C., Malnasi-Csizmadia, A and Sellers, J.R. Mechanism of action of Blebbistatin inhibition of Myosin II. J. Biol. Chem. 279(34) 35557 (2004)

Poinclouxa,R., Collina, O., Lizárragaa F., Romaoa, M., Debraye, M., Piela, M. and Chavrier, P. Contractility of the cell rear drives invasion of breast tumor cells in 3D Matrigel. PNAS 108 (5):1943 (2011)

Fischer, K.E., Pop, A., Koh, W., Anthis, N.J., Saunders,W.B. and Davis, G.E. Tumor cell invasion of collagen matrices requires coordinate lipid agonistinduced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling. Mol Cancer. 5:69 (2006)

Kinetic Chemotaxis Assays Powerful assays for quantification and visualization of chemotactic cell migration and invasion

Introduction

IncuCyte® Chemotaxis Assay at a Glance

Chemotactic migration and invasion play an essential role in many normal and pathological processes including immune response, cell differentiation and tumor metastasis. Methods to study factors that regulate the directional migration of cells typically employ modifications of Boyden chamber assays. Boyden chamber assays consist of an upper and lower chamber separated by a porous filter, allowing for the diffusion of a chemotactic agent. Although these assays offer insight into the chemotactic response of cells, they have inherent drawbacks including the inability to visualize active cell migration and the requirement for a large number of cells, typically 50,000 to 100,000 cells per well. Most importantly, perhaps, Boyden chamber assays “only afford the measurement of cell migration at a single time point, and as the kinetics of cell migration vary markedly depending on the cell type studied and the chemoattractant used, this limits the utility of this type of assay.”1

The IncuCyte® ClearView chemotaxis cell migration assay is conducted in a 96-well migration plate consisting of an optically clear membrane insert and reservoir that allows for direct visualization of cell migration and morphological changes using phase contrast and/or fluorescent imaging. The plate is placed in the IncuCyte® instrument, a fully automated live-cell analysis system with integrated image analysis tools that is placed inside a standard tissue culture incubator. Integrated metrics precisely quantify the chemotactic response using 1,000 to 5,000 cells per well, a significant advantage when using rare primary hematopoietic cells from the blood. This assay method is sensitive to surface-integrin signaling, and sustains a linear gradient over several days. The result is 96 wells of reproducible kinetic data supported by images and movies, requiring very few cells and minimal hands-on time.

In this chapter, we will review live-cell chemotatic cell migration and invasion assays, which accurately measure and visualize cell movement in response to a chemical gradient. This approach allows for the assay to be completed in a physiologically relevant environment, supports a stable gradient, and automatically analyzes images, alleviating technically challenging and cumbersome quantification steps in traditional approaches.

Live-Cell Analysis Handbook — Second Edition

Kinetic Chemotaxis Assays

Shortcomings of Traditional Assays

Live-Cell Imaging and Analysis Approaches

• Inability to visualize active cell migration.

• Real-time, direct visualization and automated analysis of chemotactic migration, invasion in a 96-well well assay format.

• Requirement for a large number of cells. typically 50,000 to 100,000 cells per well. • Measurement of cell migration at a single time point.

• Reproducible kinetic data supported by images and movies. • Requires only 1,000 to 5,000 cells per well, a significant advantage when using rare primary hematopoietic cells from the blood. • Assay is sensitive to surface-integrin signaling allowing the study of migration on biologically-relevant surfaces.

Cancer cell chemotactic migration — adherent cells The pharmacological effect of traditional signaling pathway inhibitors on the directed migration of HT-1080 fibrosarcoma cells toward fetal bovine serum (FBS) was investigated (Figure 2). We observed concentration-dependent inhibition of directed cell migration with each of the three inhibitors tested: KU0063794 (Akt pathway), U0126 (MEK/ERK MAPK pathway), and Wortmannin (PI3K pathway).

• Sustains a linear gradient over several days. • Measure label-free or labeled cell migration without fixing, staining or cell scraping steps. • Flexibility to study adherent and non-adherent cell chemotaxis, in mono- or coculture, with or without fluorescent labels.

Sample Results

Figure 2. Inhibition of HT-1080 cancer cell migration. This 96well microplate graph illustrates the kinetic measurement of HT-1080 cell migration toward 10% FBS in each well of the ClearView 96-well chemotaxis plate. Plotted in each well is the cell area on the bottom of the membrane (y-axis) over the course of a 48-hour assay (x-axis). U0126, KU0063974 or Wortmannin were added to 1,000 HT-1080 cells in the upper chamber and incubated at 37°C for approximately 30 minutes prior to exposing the cells to chemoattractant in the lower chamber. The concentration of inhibitors decreases from the top of the plate to the bottom. Positive (with chemoattractant) and negative (without chemoattractant) controls are located in the last row. These data were collected over a 48-hour period at 1-hour intervals.

Top

Automated analysis and visualization Whole-well images of cells on both the bottom and the top of the IncuCyte® ClearView plate membrane are captured at userdefined intervals. All images are processed using automated algorithms to quantify cell area on each side of the membrane (Figure 1). Directed cell migration can be reported as either an increase in area on the bottom side of the membrane for adherent cells, or a decrease in area on the top side of the membrane for non-adherent cells that migrate down the pore and fall off of the membrane.

Cancer cell chemotactic invasion Bottom

Directed cell invasion was studied by embedding HT-1080 cells in an extracellular biomatrix, and investigating the ability to specifically inhibit invasion using GM6001. Side-by side migration and invasion assays were performed (prepared by embedding 1,000 HT-1080 cells per well in 1 mg/mL Cultrex® Rat Tail Collagen 1) in ClearView plates (Figure 3) The biomatrix:cell layer was overlaid with three-fold serial dilutions of GM6001,a

broad spectrum matrix metalloproteinase (MMP) inhibitor. After compound addition, 10% FBS was added to the reservoir wells and measurements of bottom-side nuclear counts were plotted over 72 hours. Data shows specific GM6001 concentration-dependent inhibition of HT-1080 invasion and no effect on the migratory response of HT-1080 cells toward 10% FBS.

Phase

Figure 1. Chemotaxis quantification. HT-1080 fibrosarcoma cells were plated in the top chamber of the ClearView 96-well cell migration plate at a density of 1000 cells/well. 10% FBS was added to the bottom chamber as a chemoattractant. Images represent the top and bottom side of the membrane at the 36-hour time point. Automated image processing separates cells located on the top (outlined in yellow) and the bottom (outlined in blue) surface of the membrane. Pores are outlined in orange. Images are processed as they are acquired, and data can be plotted in real time. 46

Figure 3. Effect of GM6001 on migration and invasion of HT-1080 cells in a collagen invasion assay. HT-1080 cells were plated at a density of 1,000 cells per well in assay medium (A, migration assay) or embedded in 1 mg/mL neutralized Cultrex® Rat Tail Collagen 1 (B, invasion assay). Serial dilutions of GM6001 were added to the cells in the migration assay or overlaid on the biomatrix:cell layer of the invasion assay at indicated concentrations prior to exposing them to 10% FBS. Analysis of pharmacological response was performed at t=72 hr. Inhibition curve for GM6001 indicating IC50 in the invasion assay (C). Data were collected over a 72-hour period at 2-hour intervals. Each data point represents mean ± SEM, n=4. 47

Live-Cell Analysis Handbook — Second Edition

Kinetic Chemotaxis Assays

Immune cell chemotaxis – non-adherent cells In a model of immune cell chemotaxis, the response of CD3/CD28 activated T cells toward two chemoattractants was investigated: CXCL11 and CXCL12 (aka SDF-1a), ligands for CXCR3 and CXCR4, respectively (Figure 4)2. By measuring the loss of cell area on the top of the membrane, we show that activated T cells migrate toward both CXCL11 and CXCL12. Results show the selective CXCR4 antagonist, AMD3100, inhibits chemotaxis toward CXCL12 (IC50 = 279 nM), with no effect on CXCL11-mediated chemotaxis. This experiment was completed on an ICAM-1–coated surface.

Interestingly, successful measurements of T cell chemotaxis were also made on fibronectin and Matrigel®/FBS–coated surfaces, while T cells did not migrate on an uncoated surface. This suggests that interactions between integrins and/or receptors on the cell surface and the substrate play a fundamental role in T cell chemotaxis in this assay. This was not the case when tested in a traditional Boyden chamber, where the surface coating was not required (data not shown).

Figure 5. Morphological images. Phase-contrast images of neutrophils isolated in RPMI + 0.5% BSA (A) versus isolation in RPMI + 0.5% HSA (B), seeded on ClearView membranes coated with 50 µg/mL Matrigel + 10% FBS, showing morphological differences in response to 1 µM C5a (t=20 minutes). This difference in phenotype was also observed in neutrophils that were exposed to the chemoattractant IL-8.

Intra- and inter-plate reproducibility measurements

Figure 4. T cell chemotaxis toward CXCL11 and CXCL12 (SDF-1a). T-cells were plated at a density of 5,000 cells per well in the upper chamber of an ClearView 96-well chemotaxis plate coated with ICAM. AMD3100 was added to the cells at indicated concentrations and incubated at room temperature for approximately 1 hour prior to exposing the cells to chemoattractant gradient. CXC11 or SDF-1a was added to the reservoir plate at 100 nM or 50 nM, respectively, based on EC50 values obtained in agonist curve experiments (data not shown). Analysis of pharmacological response was performed at t=30 hr. Data were collected over a 30-hour period at 2-hour intervals. Each data point represents mean ± SEM, n=3.

To assess assay reproducibility and precision of the IncuCyte chemotaxis assay, a series of four independent experiments using Jurkat (non-adherent) T cells was performed (Figure 6). Cells were plated at a density of 5,000 cells per well. Two-fold dilutions of CXCL12 were made across columns, and measurements of total phase object area normalized to the initial value were plotted

over a 30-hour time course. Highly reproducible well-to-well kinetic measurements, with an average intra-assay CV of 6.3%, were observed. Similar results were obtained when measuring the directed migration of HT-1080 cells toward FBS and neutrophils toward IL-8, C5a and fMLP (data not shown).

Surface contact-mediated migration The low pore density of the ClearView membrane ensures that cells must migrate across the biologically relevant surface towards the chemoattractant. Neutrophils seeded on an uncoated ClearView membrane were unable to migrate towards the chemoattractants IL-8 and fMLP; however, those on Matrigel®-coated membranes showed clear chemotactic profiles (data not shown). These data suggest that integrin and/or cell surface receptor interactions with the substrate play a key role in neutrophil chemotaxis in this model. In contrast, no coatings were required for neutrophil migration studies using a 96-well modified Boyden chamber assay (data not shown), suggesting that active migration of neutrophils in a Boyden chamber approach is absent. Interestingly, the assay micro-environment is critical in supporting cell motility. Neutrophils suspended in RPMI supplemented with 0.5% BSA were unable to migrate toward C5a and IL-8 chemoattractant gradients. When media was

supplemented with 0.5% HSA, however, neutrophils actively migrated toward both C5a and IL-8 (data not shown). Figure 5 shows that upon visual inspection of the wells, an observed difference in cell morphology of neutrophils isolated in RPMI + 0.5% BSA (rounded phenotype), compared to neutrophils isolated in RPMI + 0.5% HSA (activated phenotype). Furthermore, neutrophils assayed using a modified Boyden-chamber approach responded to both chemoattractants (data not shown), showing no sensitivity to the different albumins present in the assay media. Together, these quantitative and qualitative data suggest that the interaction of cell integrins with the substrate and the overall assay micro-environment are a crucial component in the IncuCyte chemotaxis cell migration assay.

Figure 6. Precision and reproducibility of directed migration of Jurkat T cells toward CXCL12. Jurkat cells were plated at a density of 5,000 cells per well in the upper chamber of the ClearView chemotaxis cell migration plate. Two-fold dilutions of SDF-1a (n=8 per concentration) were added to the reservoir.

Stable gradient A long-term, stable gradient is required to support chemotaxis over longer periods of time, and to increase cell participation rates. In an experiment designed to evaluate the stability of a chemoattractant gradient in the ClearView 96-Well chemotaxis plate and a 96-well modified Boyden-chamber assay, diffusion of a 10,000 kD dextran fluorescently labeled with Alexa Fluor® 594 was monitored over a 72-hour time course in each consumable. As shown in Figure 7, more than 50% of the gradient dissipates in the 96-well traditional Boyden-chamber plate within the first

four hours. In contrast, >80% of the gradient remains intact in the ClearView plate at 72 hours. In a biological test of gradient stability, the HT-1080 cells migrated directionally toward FBS with equal rates in gradients that had been pre-established for 24, 48, and 72 hours, indicating a stable gradient across the membrane (Figure 7). These rates were slightly faster than those for cells migrating toward a gradient that had not been preestablished. This is likely a result of increased cell health when serum-starved cells are plated in established gradients. 49

Live-Cell Analysis Handbook — Second Edition A

Figure 7. Evaluation of ClearView chemotaxis assay gradient. (A) 10,000 kDa dextran, labeled with Alexa Fluor® 594, was added to the reservoir plates for the ClearView and 96-well modified Boyden-chamber at a concentration of 10 µM to establish gradients over 72, 48, 24 and t=0 hours. Measurements of diffusion were made by sampling the insert wells on both plates and measuring fluorescent intensity on a microplate reader. Each data point represents mean ± SEM, N=3. (B). An FBS gradient was established over 72, 48, 24 and t=0 hours in separate wells. Serum starved NucLight Red HT-1080 cells were added at a density of 500 cells per insert well to the established gradients and FBS-induced chemotactic response was measured. Each data point represents mean ± SEM, N=3.

Conclusions

References

The IncuCyte chemotaxis assay is a quantitative and reproducible approach for measuring chemotaxis in both adherent and nonadherent cell types. This assay format allows for the kinetic detection of cell migration toward chemotactic gradients on a physiologically relevant substrate, with movies and images that support quantitative measurements and provide associated morphological and phenotypic insights.

Taylor, L., Brodermann, M., McCaffary, D., Iqbal, A. J., and Greaves, D. R: Netrin-1 reduces monocyte and macrophage chemotaxis towards complement component C5a. PLOS PLoS ONE (2016) 11(8): e0160685.

Kawaguchi A, Orba Y et al: Inhibition of the SDF-1a-CXCR4 axis by the CXCR4 antagonist AMD3100 suppreses the migration of cultured cells from ATL patients and murine lymphoblastoid cells from HTLV-U Tax transgenic mice. Blood 2009, 114: 2961-2968.

Key features demonstrated in the IncuCyte chemotaxis assay are: •• Kinetic cell migration is automatically processed on both the top and bottom side of the optically clear membrane, allowing for real-time analysis of chemotactic driven migration of adherent and non-adherent cell types. •• Non-adherent cell migration is precisely quantified in a physiologically relevant environment, while the plate stays stationary, allowing for the evaluation of rare primary hematopoietic cells from the blood. •• Cells are required to migrate across a biologically relevant surface, demonstrating the need for cell-surface interaction, providing an opportunity for evaluation of integrin receptor signaling. •• All data points can be validated by individual images or timelapse movies to confirm processing metrics, significantly enhancing the confidence in the measured response.

Kinetic Transendothelial Migration Assays Label-free, live cell imaging assay to visualize and quantify transendothelial migration Introduction

IncuCyte® Transendothelial Migration Assay at a Glance

The recruitment of leukocytes to sites of infection and their subsequent migration through the endothelium are critical steps of the immune response. This process of transendothelial migration (TEM) is essential in order for leukocytes to respond to foreign microorganisms, but if uncontrolled, can lead to autoimmune disorders such as inflammatory bowel disease and rheumatoid arthritis. Assays to evaluate the extravasation of leukocytes are also essential for the study of host-defense response and inflammatory disorders.

The IncuCyte® Chemotaxis transendothelial migration assay is a fully automated solution to quantify directional leukocyte migration across an endothelial monolayer using kinetic, imagebased measurements. This label-free assay is suitable for testing multiple cell type pairings, evaluation of immunomodulation with blocking antibodies or inhibitors, along with the associated ability to assess monolayer integrity throughout the assay.

In this chapter, we will review live-cell transendothelial cell migration assay, which accurately quantifies directional leukocyte migration across an endothelial monolayer in real time. This approach allows for the assay to be completed in a physiologically relevant environment and automatically analyzes images, alleviating technically challenging and cumbersome quantification steps in traditional approaches.

The transendothelial migration assay is conducted in a 96well IncuCyte® ClearView Chemotaxis plate consisting of an optically clear membrane insert and reservoir that allows for direct visualization of leukocyte migration across an endothelial monolayer. The plate is placed in the IncuCyte® instrument, a fully automated live-cell analysis system with integrated image analysis tools, thus eliminating laborious endpoint analysis. Kinetic data of the 96-well plate is supported by images that allow for visualization of endothelial monolayer integrity and leukocyte diapedesis.

Shortcomings of Traditional Assays

Live-Cell Imaging and Analysis Approaches

• Standard Boyden chamber surfaces are not easily amenable to imaging.

• Acquisition of high-definition, phase contrast images enable verification of intact endothelium throughout the experiment.

• Require use of intrinsically toxic dyes.

• Eliminates use of intrinsically toxic dyes.

• Requires fixing, staining and cell scraping steps.

• Fixing, staining and cell scraping steps not required. • Quantitate cells on top and bottom of the membrane to confirm movement.

Live-Cell Analysis Handbook — Second Edition

Kinetic Transendothelial Migration Assays

Sample Results

Real-time kinetic quantitation of label-free TEM

Assessment of monolayer integrity and visualization of TEM

CD3/CD28 Dynabead activated primary T-cells were seeded on a HUVEC monolayer cultured on fibronectin in IncuCyte ClearView chemotaxis plates. Live cell images were captured at regular time intervals, and directed transendothelial migration toward CXCL12 (SDF-1a) was quantified using image analysis algorithms. Significant transendothelial migration, or diapedesis, was observed in response to increasing concentrations of SDF-1a, and could be completely inhibited using high concentrations of AMD3100, a selective inhibitor of CXCR4 and CXCL12 (SDF-1a) mediated chemotaxis.

Human umbilical vein endothelial cells (HUVECs) were grown to confluence on top of fibronectin, a physiologically relevant basement membrane. Integrity of the monolayer was evaluated both prior to leukocyte addition, using electrical resistance measurements as well as staining for E-Selectin, and after leukocyte addition by assessing phase contrast images. HUVECs were seeded at 6,000 cells per ClearView insert and allowed to form a monolayer for approximately 24 hrs. Cells were permeabilized and fixed, and VE-cadherin adhesion junctions (green) were visualized from assembled 10x Z-stack confocal images; dashed circles mark membrane pore locations (Figure 1A). Resistance measurements of HUVEC monolayers cultured on a supporting matrix of fibronectin was measured using a digital

multimeter. HUVEC monolayers were grown for 24 hours in EGM2, then growth medium was removed and 60µL of DPBS-/- was placed into the insert wells and 200 µL into the reservoir wells. The average resistance for inserts containing a monolayer was 31.9±1.7 compared to wells without cells14.9±0.9 (N=9 per condition), indicative of monolayer formation over the insert pores (Figure 1B). Label-free CD3/CD28 Dynabead activated primary T cells were added to the endothelial monolayer cultured on fibronectin in the presence or absence of migration modulators. Images were acquired every minute (Figure 2) using the IncuCyte system’s 10x objective. Yellow and orange arrows indicate leukocytes moving between HUVEC cells and eventually down the pores (blue circles) of the ClearView insert.

Whole-well, phase-contrast images represent the top-side of the membrane at t=0 hour time point (Figure 3A). Automated image

Figure 3. Data quantification and analysis. Whole-well, phase-contrast images represent the top-side of the membrane at t=0 hour time point (A). Automated image processing separates T cells (outlined in yellow) from the HUVEC monolayer (B).

The insert containing T cell:HUVEC monolayer co-cultures was exposed to 3-fold decreasing concentrations of CXCL12 (SDF-1a) (Figure 4A). Images were acquired every 30 minutes and phase analysis was performed. Analysis of pharmacological response was performed at t=6hr; each data point represents mean ±SEM, N=4 (Figure 4B).

Figure 1. Assessment of monolayer integrity. VE-cadherin adhesion junctions (green) were visualized (A) and monolayer formation confirmed via measurement of resistance across the inserts (B).

Figure 2. Visualization of leukocyte extravasation.

processing separates T cells (outlined in yellow) from the HUVEC monolayer (Figure 3B). Images are processed as they are acquired, and data can be plotted in real time as a decrease in area on the top side of the membrane for leukocytes that extravasate the endothelial monolayer and down the pore.

Figure 4. Primary T cells extravasation toward CXCL12. CXCL12 (SDF-1a) induced TEM (A). Pharmacological response of T cell migration (B). 53

Live-Cell Analysis Handbook — Second Edition

Inhibition of leukocyte extravasation For AMD3100 (CXCR4 receptor antagonist) and BIRT377 (allosteric modulator of LFA- 1) inhibition studies, CD3/CD28 activated T-cells were pre-treated for 1 hour at 37°C at indicated inhibitor concentrations, then seeded onto HUVEC monolayers and exposed to 100nM SDF-1a (Figure 5A and C). Analysis of AMD3100 pharmacological response was performed at t=6 hr. Each data point represents mean ± SEM, N=4 (Figure 5B). For antibody inhibition, HUVEC monolayers cultured overnight on fibronectin were ± pre-treated with neutralizing ICAM-1 for 1 hour at 37°C (Figure 5D). CD3/CD28 Dynabead activated T cells

were then plated at a density of 5,000 cells per well on to a HUVEC monolayer then exposed to 100nM SDF-1a (EC80concentration). Images were collected every hour over a 12 hour period and phase analysis performed. The observed increase in total cell area is due to proliferation of C3/CD28 activated T cells. The inability to fully inhibit TEM via BIRT 377 and neutralizing ICAM-1 is believed to be caused by the centrifugation step, bringing the T cells to the HUVEC monolayer, thus bypassing the cell adhesion step.

Figure 5. Treatment effects on leukocyte transendothelial migration. Inhibition of CXCL12 (SDF-1a) driven T cell migration by AMD3100 (A), BIRT 377 (C) and neutralizing ICAM-1. Analysis of AMD3100 pharmacological response (B). Migration across the endothelial layer was at least partially dependent on ICAM-1, as neutralizing ICAM-1 antibodies, and treatments with an allosteric inhibitor of LFA-1, BIRT377, significantly inhibited SDF-1a mediated diapedesis of T cells.

Conclusions The IncuCyte® chemotaxis transendothelial assay can be used for real-time visualization and automated analysis of transendothelial migration in a 96-well format, in real-time. •• Acquisition of high-definition, phase contrast images ensure endothelium integrity throughout the experiment. •• Measurement of leukocyte extravasation is automatically processed without use of intrinsically toxic dyes, fixing, staining and cell scraping steps. •• All data points can be validated by individual images or time-lapse movies to confirm processing metrics, significantly enhancing the confidence in the measured response. 54

Kinetic Assays for Quantifying Protein Dynamics Complementary, live-cell solutions for studying protein dynamics

Introduction Proteins – the building blocks of the cell – are required for virtually every biological process, from cell function, to structure and regulation. The study of proteins – how they move, interact and communicate with each other – is thus essential for our understanding of health and disease. This is particularly true of proteins on the surface of cells involved in cell signaling and ligand binding, which requires the interaction of both extracellular proteins, such as cytokines and hormones, and membrane proteins. Such interactions are often the drivers of disease processes or the basis of drug mechanism of action. Antigen-mediated antibody internalization, for example, plays an important role in several antibody-based therapeutics and in differentiation-induced protein expression changes. However, despite their importance, our ability to analyze these processes and link their properties to cell function has been hindered by the methodologies available to study them. Current methodologies for analysis of membrane proteins and their receptor function are limited to single time point analyses, not taking into account the dynamic changes in protein expression due to protein turnover or in response to biochemical cues. While traditional protein assays have provided insights into localization, binding and protein-protein interactions; these single snapshots in time cannot follow the dynamics of proteins in living cells under physiological conditions and therefore are lacking in physiological context. IncuCyte’s live-cell analysis system and antibody labeling reagents enable a new assay approach that delivers spatial and temporal information at scale in living cells. The unique antibody reagents can be mixed with any Fc-containing antibody, applied directly to cells and then monitored under physiological conditions for hours or days. The real-time image acquisition and analysis means it is possible to quantitatively link the dynamics of a target protein

with cell morphology, proliferation and function. The protocol is simple despite the richness of data and insight generated, involving a single antibody-labeling step followed by direct addition to cells, thereby removing the need for laborious labeling and blocking procedures and wash steps that can result in unwanted cellular changes and misleading results. IncuCyte® FabFluor antibody labeling reagents are highly specific and sensitive, amenable to both adherent and non-adherent cell models. The data below exemplifies how the assays can be used to study long-term protein dynamics and their relationship to cell function, morphology, as well as cell-cell interactions.

Live-cell imaging and analysis approaches to studying protein dynamics Fluorescently labeled antibodies are widely used as markers for visualizing cell surface protein expression, receptor interactions and antibody internalization. To build on existing methodologies for studying protein expression, a novel strategy was developed based on fluorescently labeled antibody fragments (Fabs) that target the Fc region of any mouse, human or rat antibody. Two types of reagents have been developed, differing in the type of fluorescent molecule conjugated to the Fab and thereby enabling two different applications, antibody internalization and live-cell immunocytochemistry. These two reagents, when combined with the IncuCyte® Live-Cell Analysis System, provide an integrated approach to studying protein dynamics in real-time and under physiological conditions.

Live-Cell Analysis Handbook — Second Edition

How Live-Cell Protein Assays Work Antibody internalization assays

Live-cell immunocytochemistry

In the antibody internalization assay, a pH-sensitive antibody labeling reagent, IncuCyte® FabFluor-pH Red, allows the specific detection of antibodies as they are internalized into the acidic conditions of endosomes and lysosomes, enabling real-time, kinetic monitoring over the full time-course of internalization.

To detect cell surface markers, IncuCyte® FabFluor-488, a Fab conjugated to a continuously fluorescing molecule at physiological pH, is utilized for antibody labeling. Addition of the IncuCyte® FabFluor-antibody complex to living cells enables kinetic detection of cell surface markers, making it possible to track the movement and interaction of cell subsets, reveal the dynamics of their interactions, and link time- and concentrationdependent changes in cell surface protein expression to other morphological and phenotypic parameters.

References 1.

Cohen AS, Khalil FK, Welsh EA, Schabath MB, et al. Cell-surface marker discovery for lung cancer. Oncotarget 2017, 8(69): 113373-402.

Gundry RL, Boheler KR, Van Eyk JE, and Wollscheid B. A novel role for proteomics in the discovery of cell-surface markers on stem cells: Scratching the surface. Proteomics Clin. Appl 2008, 2(): 892-903.

Macher BA and Yen TY. Proteins at membrane surfaces – a review of approaches. Mol BioSys 2007, 3(): 705-13.

Roesli C, Borgia B, Schliemann C, Gunther M, et al. Comparative analysis of the membrane proteome of closely related metastatic and nonmetastatic tumor cells. Cancer Res 2009, 69(13): 5406-14.

Kuhlmann L, Cummins E, Samudio I, and Kislinger T. Cell-surface proteomics for the identification of novel therapeutic targets in cancer. Expert Review of Proteomics 2018, 15(3): 259-75.

Ritchie M, Tchistiakova L, and Scott N. Implications of receptor-mediated endocytosis and intracellular trafficking dynamics in the development of antibody drug conjugates. mAb 2013, 5(1): 13-21.

Beck A, Haeuw JF, Wurch T, Goetsch L, et al. The next generation of antibody-drug conjugates comes of age. Discov Med 2010, 10(53): 329-39.

Adams GP and Weiner LM. Monoclonal antibody therapy of cancer. Nat Biotechnol 2005, 23(9): 1147-57.

Liao-Chan S, Daine-Matsuoka B, Heald N, Wong T, et al. Quantitative Assessment of Antibody Internalization with Novel Monoclonal Antibodies against Alexa Fluorophores. PLoS ONE 2015, 10(4): e0124708.

10. Kuo S, Alfano RW, Frankel E and Liu J. Antibody Internalization after Cell Surface Antigen Binding is Critical for Immunotoxin Development. Bioconjugate Chem 2009, 20(10): 1975-82.

Kinetic Antibody Internalization Assays Visualization and quantification of antibody internalization, in real time

Introduction The growing focus on antibodies as therapeutics makes it essential to be able to study their dynamics in a realistic cell environment. Antibodies are used both as therapeutic agents that themselves block the activity of certain molecules, or for the targeted delivery of treatments to sites or cells of interest. This includes the delivery of highly toxic drugs to cancer cells via antibody drug conjugates (ADCs), removal or degradation of surface receptors from cancer cells (i.e. EGFR), and antibody immunotherapies used to identify tumor cells for immune cell killing (i.e. ADCC or ADCP). Each of these therapeutic strategies hold the promise of increased efficacy and reduced side effects, and each requires a series of antibody features, for example, to enable maintenance on the cell surface for identification of tumor cells, or for rapid internalization when delivering ADCs. To realize their potential, it is important to understand the uptake profile and clearance of antibody candidates, and to be able to measure and optimize functional responses to antibodies for optimal antibody engineering and internalization characteristics. For example, pinocytosis, which is one of the main elimination routes of antibodies, requires antibody optimization for qualitative pharmacokinetic measurements during therapeutic antibody development. Current methods for measuring antibody internalization have several limitations. These include the requirement to label each antibody, conduct multiple washing steps, and the ability to only do singletime point analysis. This can lead to unnecessary and labor-intensive processes, loss of cells through washing, and missed and incorrect measurements due to limitations of single time-point analysis.

There is an urgent need to have an efficient, affordable method that can be used throughout the entire antibody screening and functional characterization process to fully and rapidly quantify antibody internalization using real time live-cell analysis. In this chapter, we will illustrate how novel pH-sensitive Fctargeting antibody labeling reagents allow for real-time rapid, kinetic, high-throughput analysis of antibody internalization in living cells. This is an ideal tool for optimal antibody engineering early in the biologic discovery process.

IncuCyte® FabFluor antibody internalization assays at a glance The IncuCyte® antibody internalization assay uses IncuCyte® FabFluor-pH Red reagent, a novel pH-sensitive Fc-targeting antibody fragment, isotype-matched to the antibody of interest. The IncuCyte® FabFluor-pH Red reagent and antibody of interest are mixed in a one-step, no wash, labeling protocol. At pH 7.0, the FabFluor-Ab complex has little or no fluorescence. When labeled antibodies are added to cells, a fluorogenic signal is seen as the antibody is internalized and processed in acidic (pH 4.55.5) lysosomes and endosomes. The full time-course of antibody internalization can then be followed with the IncuCyte Live-Cell Analysis System for real-time analysis of internalization rates. An overview of the workflow is shown on the next page. (Figure 1)

Shortcomings of Traditional Assays

Live-Cell Imaging and Analysis Approaches

• Requires labor-intensive labeling of each antibody.

• Rapid, single-step labeling allows efficient testing of panels of antibodies.

• Only provides single-time point analysis. • Low throughput due to combination of labeling approach and end-point analysis.

• Real-time, kinetic measurements of antibody responses. • High-throughput, reproducible antibody screening validated with individual images.

Live-Cell Analysis Handbook — Second Edition 1

Seed cells

Cell Seeding Seed cells (50 µL/well, 5,000-30,000 cells/well), into 96-well plate and leave to adhere (2-24 h, depending on cell type).

Figure

Label test antibody

Labeling of Test Antibody with IncuCyte® FabFluor-pH Red Reagent Mix antibody and FabFluor Reagent at a molar ratio of 1:3 in media, 2x final assay concentration. Incubate for 15 minutes to allow conjugation.

Kinetic Antibody Internalization Assays 3

Add to cells

Live-cell fluorescent imaging

IncuCyte® FabFluorlabeled Antibody Addition

Automated Imaging and Quantitative Analysis

Add antibody-FabFluor mix (50 µL/well) to cell plate.

Capture images every 15-30 minutes (10x or 20x) in IncuCyte® for 24-48 hours. Analyze using integrated software.

Specific detection and visualization of internalization The IncuCyte® analysis software robustly detects when an IncuCyte® FabFluor-pH Red labeled antibody has been internalized. Duallabeling experiments were conducted in HT1080 fibrosarcoma cells using a lysosomal marker (LysoSensor® Green, Thermo Scientific) and an IncuCyte® FabFluor-pH Red-labeled antiCD71 antibody (Figure 3). After three hours, a strong co-incident signal was measured: 74% of the red anti-CD71 signal was colocalized with green, confirming that the labeled antibody was internalized in the lysosome.

1. Overview of IncuCyte® antibody internalization assay

Sample Results

Antibody internalization with IncuCyte® FabFluor-pH Red reagent Antibody internalization signal increases with cell number, but IncuCyte® antibody labels can distinguish the true internalization rate signal from that associated with cell proliferation by virtue of collecting both biochemical and morphological data simultaneously. AntiCD71 internalization (red fluorescence area) was normalized to the total cell area (phase confluence), the internalization signals over time between different plates were highly similar. This normalization method helps to minimize the impact of variation in cell number between plates and reveals the true internalization rate of your antibody of interest.

Figure 2. Antibody internalization response is cell number dependent. An increasing density of HT1080 cells were seeded (1-20K/well) and treated with IncuCyte® FabFluor-pH labeled α-CD71 (4 µg/mL). HD phase and red fluorescence mages (10X) were captured every 30 min over 12 h. The time-course of red object area data demonstrates an increasing internalization signal with increasing cell number (A and B). When the red object signal is normalized for phase area, it is clear the internalization response size is depending on cell number (C and D). All data shown as a mean of 3 wells ± SEM, bar graphs shown as area under the curve (AUC) calculated from time-course data. 58

Figure 3. Colocalization of IncuCyte® FabFluor labeled α-CD71 and Lyso Sensor® Green in HT1080 cells. Internalization of IncuCyte® FabFluor labeled α-CD71 (4 μg/mL) was established for 3 h in HT1080 cells before addition of LysoSensor® Green DND-189 (Thermo, 0.25 μM). Images show individual LysoSensor® Green and FabFluor labeled α-CD71 red signal (A and B), co-localization of red and green signals (C), and the co-localized analysis mask shown in yellow (D). (E) IncuCyte analysis of the coincidence of the red and green fluorescence confirms co-localization of 74% of the red signal with the green signal. Images captured at 20x magnification, 30 min post LysoSensor® addition, data shown as mean of 4 wells ± SEM.

Live-Cell Analysis Handbook — Second Edition

Kinetic Antibody Internalization Assays

Quantitative Pharmacological Analysis Applicability across different cell types and antibodies To demonstrate the broad application and specificity of the method, internalization was assessed for a range of test antibodies targeted against specific CD markers expressed in different cell lines (Figure 5). Anti-CD20 was internalized in the B-cell line Raji, but not Jurkat, a T-cell line. Conversely, anti-CD3 was internalized in Jurkat but not Raji. Antibodies to CD71 and CD45,

Pharmacological, kinetic quantification of antibody internalization general lymphocyte markers, were internalized in both cell types. Importantly, IgG was not internalized in either. These data are in alignment with the known CD surface marker expression of these cell lines, and provide strong confidence in the signal specificity and generic utility of the method.

To illustrate the quantitative nature of the method and suitability for analyzing therapeutic antibodies, we sought to determine EC50 values for the internalization of Herceptin (Trastuzumab) and Rituxan (Rituximab), two clinically used monoclonal antibodies. After labeling of each antibody with the FabFluor reagent, the antibody was serially diluted (1:2) prior to addition to the cells to enable construction of a concentration-response curve. Handling the labeled antibody this way is good practice to eliminate any variation in Fab labeling efficiency which

could occur across the concentration range. In BT-474 Her2positive breast carcinoma cells, clear time and concentration dependent internalization of Herceptin was observed over 48 h. From an area under the time-course (AUC) analysis, the EC50 value for internalization was 323 ng/mL ≡ 2.1 nM, Figure 6). In Raji cells, the EC50 value for Rituxan was 426 ng/mL ≡ 2.6 nM, Figure 7). These EC50 values are similar to the known KD values for Herceptin and Rituxan for their target receptors (both approximately 5 nM).

C A

Figure 6. Quantitative pharmacological analysis of IncuCyte® FabFluor labeled Herceptin. BT-474 Her2-positive cells were treated with increasing concentrations of FabFluor labeled Herceptin. The time course graph displays an increase normalized red area over time with increasing Herceptin concentrations (A). Area under the curve analysis of this response displays a clear concentration dependent response with an EC50 of 323 ng/mL (B). All data shown as a mean of 3 wells ± SEM, time course data shown as normalized red area.

Figure 5. Internalization of CD surface marker targeted antibodies in lymphocytic cell lines. Jurkat (T cell-like) and Raji (B cell-like) cells (30 K/well) were treated with different IncuCyte® FabFluor labeled antibodies (4 μg/mL). HD phase and red Images were captured every 30 minutes using a 20x objective over 12 h. Time course data (A and B) and area under the curve (AUC, C) analysis demonstrates the response profile in both cell lines. All data shown as a mean of at least 4 wells ± SEM, time course data shown as normalized red area.

Figure 7. Quantitative pharmacological analysis of IncuCyte® FabFluor labeled Rituxan. Raji cells were treated with increasing concentrations of FabFluor labeled Rituxan. The time course graph displays an increase normalized red area over time with increasing Rituxan concentrations (A). Area under the curve analysis of this response displays a clear concentration dependent response with an EC50 of 426 ng/mL (B). All data shown as a mean of 3 wells SEM, time course data shown as normalized red area.

Live-Cell Analysis Handbook — Second Edition

Kinetic Antibody Internalization Assays

Comparison of multiple test antibodies for high-throughput screening

Conclusions

The features of the IncuCyte and FabFluor solution are such that it should be facile to parallel label many antibodies and compare their internalization. To validate this we took 6 different commercially available anti-CD71 antibodies and compared their internalization properties head to head. The antibodies were plated in 96-well plates and labeled in full media with the IncuCyte® FabFluor labeling reagent. Serial dilutions were performed in full media (8 point, 1:2). Labeled IgG and FabFluor alone were added to control wells. Labeled antibodies were then added to pre-plated HT1080 cells and monitored for internalization for 12 h.

The key features of the approach described in this application note are:

Of the 6 antibodies, 3 (Ab 1a, Ab2 and Ab 1b) produced large internalization signals and were detected at low concentrations

(<0.05 ug ml-1). Reassuringly, Ab 1a and Ab 1b were the same antibody clone from different suppliers, and gave similar internalization responses. Abs 3, 4, and 5 were internalized more weakly and only at higher concentrations (Figure 8). From the control responses, a mean Z’ value of 0.82 was determined (2 plates 0.75, 0.87) indicating a microplate assay with high robustness. These data confirm the suitability of the method for comparing the internalization of multiple antibodies at a single target, and illustrate that the internalization profile is a property of the antibody per se. Indeed, the assay precision and work flows are such that 100s of different antibodies could be compared at once and further throughput could be achieved through miniaturization to 384-well format.

AB 1a

AB 2

AB 3

controls

AB 1b

AB 4

AB 5

•• A single step labeling protocol for easily tagging antibodies of interest with an Fc-targeted Fab coupled pH-sensitive dye (IncuCyte FabFluor). The labeling method is conducted in full media and is suitable for purified antibodies and antibody supernatants. •• An automated, image-based and real time analysis method (IncuCyte) for monitoring internalization in multiple 96-well microplates at once. The format is amenable to both adherent and non-adherent cells.

Taken together, these attributes provide a simple, integrated and quantitative solution for directly studying internalization of antibodies into cells that can easily be scaled to compare many antibodies (10s-100’s) in parallel. This method enables antibody internalization measurements to be implemented at earlier stages in the biologics discovery process, and will prove valuable in efficacy, safety and pharmacokinetic optimization of novel therapeutic antibodies. In addition, the method is suited to understanding basic mechanisms of endocytosis, pinocytosis and receptor turnover where antibodies can be employed.

•• An assay system that follows the full time course of the biology and reports internalization with high specificity, sensitivity and morphological information. The use of a pH-sensitive dye provides for low background signal and obviates the need to separate out fluorescence arising from antibody on the cell surface or in bulk solution.

controls

Figure 8. Screening test Abs for internalization. Six different CD71 antibodies including one clone from 2 different suppliers (clone 1a & 1b) were tested head to head in HT1080 cells. The antibodies were labeled with IncuCyte® FabFluor reagent prior to addition to cells and the internalization signal captured every 30 min over 12 h using a 10x magnification. Plate views taken from IncuCyte show clear positive and negative control responses in column 11 and 12 with concentration dependent responses for each antibody across two plates (A). Head to head analysis of antibody data shows a range of responses across these clones (B) control responses at 12 h display a clear positive response. All data shown as mean of 3 wells ± SEM, controls shown as mean of 8 wells.

Live-Cell Analysis Handbook — Second Edition

Live-cell Immunocytochemistry

Key advantages: • Measure surface protein expression and distribution over time using non-perturbing antibody labeling reagents in physiologically relevant conditions.

Long-term tracking and quantification of cell surface protein markers

• Associate changes in surface protein expression with cell function and morphology to reveal informative, temporal changes in cell behavior.

• Visualize and quantify cell–cell interactions over time in complex co-culture models, revealing insight into the interplay of cells. • Significantly increase productivity compared to conventional ICC by combining a rapid, single-step labeling protocol with automated acquisition and analysis. (Table 1).

Introduction Immunocytochemistry (ICC) is a powerful laboratory technique for visualizing the cellular and subcellular location of proteins using fluorescent-labeled antibodies (‘immunofluorescence’). The study of cell protein expression, and the modulation of it, is a crucial method used to understand gene expression, and ultimately the biology of the cell. With cell fixation protocols, labeled antibodies and specialized microscopes, spatial resolution down to the nanometer level can be achieved to evaluate the expression of proteins in the nucleus, specialized organelles and the cell membrane. Where conventional ICC is less useful, however, is in studying dynamic changes in protein expression over time and under physiologically relevant conditions. The technique includes a number of lengthy and time-consuming steps – fixing, washes, and staining – which can take anything between six and 24 hours. These procedures can also lead to physical loss of cells, or result in unhealthy cells that are losing their viability because of fixation and are unlikely to give an accurate picture of the biology of interest. ICC is a widely used technology for studying changes in protein surface molecules – the key communicators that dictate how a cell responds to external stimuli and changes its shape, protein expression and function in response. These cell surface molecules are the targets of many existing and future drugs and being

1 Seed cells

Cell seeding Seed cells (50 µl/well, 5-30K/well) into 96-well plate. NOTE: for non-adherent cell types, PLO coat plate prior to cell seeding.

2 Label test antibody

able to monitor their activity in real-time and in their natural environment is paramount. Consequently, there is a strong unmet need for technical solutions that can allow the tracking of protein distribution and abundance over time in living cells, and link these vto cell morphology and function. In this chapter, we illustrate how kinetic, live-cell analysis of surface protein expression using fluorescently-labeled antibodies opens up new possibilities for connecting long-term dynamic changes in protein abundance and distribution in cells with morphology and function.

IncuCyte® live-cell immunocytochemistry at a glance

Traditional Fix and Stain ICC

Suitable for

Cell surface proteins

Both intracellular and cell surface proteins

Optimized for

Protein dynamics and linking changes to function and morphology. Cell identification and cell-cell interactions in motile systems

In-depth structural/morphological analysis, subcellular distribution, organelles, protein trafficking and redistribution

Reagents

Primary antibody + IncuCyte® FabFluor-488 + Opti-Green background suppressor

F-labeled primary or secondary Ab

Hardware

IncuCyte® S3 (and IncuCyte® ZOOM)

Fluorescent microscopes, high-content imagers (IncuCyte)

Protocols

Mix and read (one hour prep time)

Fix, wash and stain (6—24 hours?)

Resolution

Cellular, up to 20x magnification

Subcellular > 100x possible with oil immersion, etc.

Cell status

Living cells in media/serum

Dead/dying cells post fixation

Imaging paradigm

Repeated imaging over several days, time-lapse movies

Single time point (“end point”)

Table 1: Comparison of IncuCyte Live Cell ICC with conventional ‘fix and stain’ ICC.

In order to measure the dynamic changes in cell surface protein expression while simultaneously monitoring morphological changes, IncuCyte® FabFluor-488 Antibody labeling reagents are combined with Fc-containing antibodies. These conjugated FabFluor-Ab complexes are then added directly to live-cells in a single-step mix and read assay. Time-lapse images of cellular fluorescence can then be gathered over hours and days, and automatically analyzed to provide an index of the levels and pattern of expression over time. An overview of the assay workflow is shown below.

3 Add incucyte opti-green

Labeling of test antibody with IncuCyte® FabFluor-488 reagent Mix antibody and FabFluor-488 reagent at a molar ratio of 1:3 in media, 3x final concentration. Incubate for 15 minutes to allow conjugation.

IncuCyte® Live-Cell ICC Approach

IncuCyte® OptiGreen background suppressor addition Add 50 µl/well, (3x final concentration).

4 Add labeled AB

IncuCyte® FabFluor-488labeled antibody addition Add antibody-FabFluor mix (50 µl/well) to cell plate. Non-adherent cells – spin plate

5 Live-cell fluorescent imaging

Automated imaging and quantitative analysis Capture images, (time span and objective depends on assay and cells type, 10x or 20x) in IncuCyte.

Figure 1. Overview of IncuCyte® live-cell immunocytochemistry workflow. 64

Live-Cell Analysis Handbook — Second Edition

Live-cell Immunocytochemistry

Sample Results

Coupling protein dynamics to morphological changes

Quantitative measurements of surface protein dynamics

Protein dynamics can also be coupled to morphological changes using IncuCyte® live-cell analysis. Here, measurements of cell surface markers were linked to morphological changes in human neuroblastoma cells (Figure 3). Here, once treated with a vitamin A

Given the current explosion of checkpoint inhibitor cancer therapies, assays that expedite further studies on the regulation of immune-cell signaling pathways in tumors are an area of significant need. The below example illustrates how dynamic changes in cell surface checkpoint proteins can be quantified in living cells in response to an inflammatory stimulus, using Programmed Death Ligand-1 (PD-L1) as an archetype (Figure 2). The IncuCyte® FabFluor-488 antibody reagent was conjugated to anti-PD-L1 and applied the mixture to MDA-MB-231 breast cancer cells. Following treatment with IFN-γ, a time- and concentrationdependent increase in PD-L1 labeling was observed over 72 hours. IFN-γ had no effect on the growth rate of MDA-MB-231, indicating that the response was a specific upregulation of PD-L1.

Day 0

Day 1

Day 2

Day 0

Day 2

Day 7

Day 14

derivative to induce differentiation, there was a clear association between the change in neural cell adhesion molecules (NCAMs) observed through live-cell ICC and the increase in neurite length over time.

Figure 3: Dynamics of neurite outgrowth and CD56 expression in atRA-differentiated SH-SY-5Y cells. Cells were incubated with FabFlour-488 conjugated anti-CD56 and treated with atRA (50μM) at t=0. Blended images show green fluorescence (CD56) and phase morphology. Neurites are masked in yellow (IncuCyte Neurotrack). The time-course of upregulation of CD56 (mean values ± SEM, 4 wells) and the relationship between CD56 and neurite outgrowth (normalized to confluency) are shown in panels A and B, respectively. In panel B, each symbol represents data taken at different time-points throughout the experiment. Note the time-dependent increase in neurite length and associated CD56 signal.

Figure 2: Upregulation of PD-L1 checkpoint protein in tumor cells in response to IFN-γ. FabFluor-488 was conjugated to anti-PD-L1 Ab (BioLegend) and added to MDA-MB-231 NucLight Red breast cancer cells in the absence and presence of IFN-γ (+ IncuCyte Opti-Green background suppressor). Quantification of the green fluorescent area shows that IFN-γ induces a time- and concentration- dependent increase in PD-L1 expression, with a mean EC50 value of 9.0 ng/mL. Values are shown are mean ± SEM, from four wells.

Coupling protein dynamics to cell function Live-cell ICC can also be used to correlate changes in morphology with function. In the below example, the differentiation of human monocytic cells into macrophage-like cells was tracked by IncuCyte® FabFluor-488 labeling of immune cell surface markers (Figure 4). It was found that only phorbol myristate acetate (PMA) produced a marked change in morphology, yielding large, flattened, adherent

‘macrophage-like’ cells. To correlate these observations with function, the ability of differentiated THP-cells to phagocytose IncuCyte® pHrodo-labeled apoptotic T cells was measured. Only those cells treated with PMA were phagocytic, illustrating that it is possible to associate protein measurements to the functional properties of cells.

Live-Cell Analysis Handbook — Second Edition

Live-cell Immunocytochemistry

Monitoring cell-cell interactions Finally, live-cell ICC was used to study the dynamic interactions of immune cells and tumor cells, in a co-culture model system (Figure 5). Close inspection of the IncuCyte® time-lapse movies revealed that individual and sometimes multiple immune cells associate with the target cell and remain attached even as the tumor cells move.

CD45

Undifferentiated

Vitamin D3 (50 nM)

Using the CD8-labeling antibody it was possible to show that a subset of CD8+ cytotoxic T-lymphocytes specifically engaged with the tumor cells, and even detected a polarity to these cells, where the CD8+ region of the effector cell appeared to contact the target.

Non-activated PBMC

PMA (100 nM)

Activated PBMC

CD8

Undifferentiated Vitamin D3

PMA

CD11b CD14 CD40 Growth inhibition Δ Morphology Phagocytosis Figure 4: Differentiation of hTHP-1 monocytes: surface marker and functional changes. hTHP-1 cells were exposed to media (undifferentiated), vitamin D3 (50 nM) or PMA (100 nM) in the presence of IncuCyte FabFluor-488 reagent complexed to CD11b, CD14 or CD40 Abs (+ IncuCyte Opti-Green). IncuCyte images (Top, 20x, every 3h) were analyzed for marker expression (green fluorescent area), normalized for confluency (Center: mean ± SEM, 4 wells). In the lower panel the phagocytic effects were determined by exposing the cells to pHrodo-labeled apoptotic Jurkat cells (1x106 per well) at 72h, and measuring red fluorescence. Note the association of the CD40 marker expression with the functional profiles (orange = +ve). 68

Figure 5: Visualization and quantification of cell-cell interactions. A549 CytoLight Red tumor cells were mixed with either pre-activated (anti-CD3/IL-2) or non-activated PBMC’s in the presence of IncuCyte FabFluor-488-α-CD45 (upper) or CD8 (lower panel). The masked image (center) illustrates the overlay (yellow) of the immune cells (green) and tumor cells (red). The overlay area metric shows the increase in interaction between the two cell types upon activation, either without (upper) or with (middle) normalization to the green area. For CD8, note the clear polarity of the protein signal in the lymphocyte, and formation of the CD8/immune cell ‘synapse’.

Live-Cell Analysis Handbook — Second Edition

Conclusions IncuCyte® live-cell Immunocytochemistry demonstrates how traditional ICC can be extended from a traditional ‘fix and stain’ approach to a method that allows for the dynamic monitoring of surface proteins in living cells over several days. Using a simple and robust labeling approach, IncuCyte® FabFluor-488 labeling reagents in conjunction with the IncuCyte® Live-Cell Analysis System, enables real-time quantification of dynamic changes in protein abundance and distribution with the ability to associate changes in cell morphology, function as well as study complex cell interactions.

•• Proximity relationships, such as those between the tumor and immune cells presented here, can be observed and quantified at throughput with unprecedented accessibility.

•• Live-cell ICC can reveal time-and concentration-dependent changes in cell surface, and can be combined with analyses of other morphological and phenotypic parameters to link surface protein expression changes with cell function over time proteins.

•• The reagent removes the need for researchers to source antibodies with appropriate fluorescent tags for their molecule of interest.

•• By labeling different cell surface markers with IncuCyte® FabFluor-488 reagent it becomes possible to track the movement and interaction of subsets of living cells, revealing the dynamics of their interactions.

•• IncuCyte® FabFluor-488 antibody labeling reagent is specific, stable under physiological conditions for long incubations, and allows image analysis without disturbing the biology, health or proliferation of cells. •• The reagent has been used successfully with a range of antibodies in a variety of cells displaying surface proteins – including immune cells, nerve cells and tumor cells.

•• The live-cell ICC method saves time – assays leverage automated acquisition and analysis of data and can be carried out simultaneously in up to six 384-well plates, without laborious fixation and washing protocols.

Kinetic Assays for Utilizing Complex Models Live-cell microplate assays for studying growth and health of three-dimensional cultures

Introduction It is well established that conventional monolayer cultures of tumor cells grown on plastic do not adequately reflect the in vivo situation.1,2 Yet, in vitro culture models of cancer cells are still an integral component of clinical drug development and for the advancement of our understanding of cancer cell biology. 3D cell culture models, although still relatively new compared to traditional 2D monolayer cultures, are increasingly adopted as advanced tools to accelerate drug discovery efforts. 3 This shift is most notable in cancer biology, immuno-oncology and hepatotoxicity, where organoids and 3D microtissues can represent more relevant biological features such as hypoxic regions or tumor-immune cell interactions.4,5 There are a multitude of approaches to generating these models and they can generally be described as scaffold-free (media-based) or scaffold-based. Scaffold-free models are easily achieved using round-bottom ultra-low attachment (ULA) microplates to promote spheroid selfassembly. These models generally yield a single tumor spheroid per well and exhibit key features of solid tumors; larger spheroids consisting of proliferating, quiescent, and necrotic zones resulting from a radial gradient of nutrients, metabolites and oxygen. Current scaffold-based models rely primarily on an extracellular matrix (ECM) such as Matrigel速 or collagen and attempt to recapitulate both physical and biochemical characteristics of the tumor microenvironment. Generally, these models employ the use of flat bottom plates and result in multiple tumor spheroids per well. Choosing a model depends on the scientific question or objective at hand. Single spheroid models may be more representative of large solid tumors and can be made with high reproducibility which

is desirable for drug testing. Multi-spheroid models capture the inherent heterogeneity of tumor cells and are considered by many to be more physiologically relevant. Employing these complex models is challenging due to inherent limitations for studying cell growth, kinetics, and other characteristics in this 3D setting. These include time-consuming, expensive or laborious workflows, labeling requirements that can affect cell biology, and single time-point or indirect read-outs that do not report the full experimental time-course and potentially miss valuable information about size or growth, or shifts in the characteristics of cell populations. The IncuCyte速 S3 Live-cell Analysis System, for both scaffold-free and scaffold-based 3D spheroid models, avoids such limitations by providing simple, cost-conscious protocols that result in 3D cultures amenable to imaging, employing either label-free techniques or use non-perturbing reagents, and enabling real-time monitoring of morphological and cell health measurements without removing precious samples from the incubator. The approach works both with single spheroids grown in round-bottomed ULA wells in 96- and 384-well formats, and multi-spheroid cultures grown on plates coated with Matrigel速 to reproduce the ECM scaffold. The IncuCyte速 approach has many advantages over traditional cell analysis methods (summarized in Table 1). In the following chapter, the assays are described in detail, demonstrating their potential as a valuable tool for insightful scientific discovery and drug screening and development.

Live-Cell Analysis Handbook — Second Edition

Kinetic Assays Utilizing Complex Models

Shortcomings of Traditional Assays

Live-Cell Imaging and Analysis Approaches

• Endpoint collection of data misses important information between imaging intervals that is needed for complete insight into biological processes.

• Continuous long term monitoring captures important kinetic information on cellular processes and morphology, providing for additional mechanistic details.

• Repeated removal of cells from incubator for lengthy image acquisition results in loss of control for O2 and CO2 and multiple environmental fluctuations. • Incorporation of labels that can perturb cells and may confound interpretation. • Additional time required for development and optimization of image acquisition parameters. • Complex image processing requires expert operation and training for data generation and analysis.

• Uninterrupted environmental control within incubator, coupled with automated image acquisition (DF Brightfield, HD Phase, and fluorescent) results in a continuous physiologically-relevant environment. • L abel-free analysis of growth and shrinkage. • Non-perturbing, optimized, validated cell health reagents for enhanced physiological relevance. • Automated acquisition and analysis of high quality, high contrast images, combined with lab-tested protocols, to quickly gather reproducible, quantitative data for pharmacological screening in 96 and 384-well formats. • Purpose built software tools and guided interface enables non-expert operators to perform image processing and generate publication-ready graphics.

References 1.

Antoni D, Burckel H, Josset E, and Noel G. Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci. Mar 11; 16(3); 5517-27 (2015)

Cui X, Hartanto Y, and Zhang H. Advances in multicellular spheroids formation. J R Soc Interface. Feb; 14(127) (2017)

Fang Y and Eglen RM. Three-Dimensional Cell Cultures in Drug Discovery and Development. SLAS Discov. Jun; 22(5); 456-472 (2017)

Costa EC et al. 3D tumor spheroids: an overview on the tools and techniques used for their analysis. Biotechnol Adv. Dec; 34(8);1427-1441 (2016)

Zanoni M. et al. 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci Rep. Jan 11;6:19103 (2016)

Recent IncuCyte® Publications 3D Killing Assays 1.

Catchpole I, et al. Engineering T-cells for adoptive cell therapy to overcome TGF-β-mediated immunosuppression in the tumour microenvironment. Annals of Oncology;28 (S11) mdx711.081, 100P (2018)

Invading cells from 3D spheroid

Table 1. Traditional vs Live-cell Analysis.

Fujita, M, Imadome, K, and Imai, T. Metabolic characterization of invaded cells of the pancreatic cancer cell line, PANC-1. Cancer Sci., 108(5); 961-971 (2018)

Tumorsphere Growth Kinetics and Cell Number How Live-Cell Spheroid Assays Work Multi-spheroid assays

Single spheroid assays

Scaffold-based, multi-spheroid assays allow for study of cell cultures in a more physiologically relevant, 3D format. These assays permit the tracking and quantification of spheroid formation, cell-cell, and cell-ECM interactions in real time inside a tissue culture incubator.

These assays provide an integrated solution to automatically track and quantify single tumor spheroid formation, growth, and health in real time with minimal environmental disturbance inside a tissue culture incubator, providing additional physiological insights.

Cazet AS, et al. Targeting stromal remodeling and cancer stem cell plasticity to overcome chemoresistance in triple negative breast cancer. Pre-print under review Cold Spring Harbor Laboratory, bioRxiv preprint (2018) URL: https://www.biorxiv.org/content/early/2017/11/08/215954

Galbraith MD, et al. CDK8 Kinase Activity Promotes Glycolysis. Cell Rep, Cell Rep, 21(6); 1495-1506, (2018)

Cesi G, et al. ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells Cancer, Mol. Cancer 16(1); 102, 42894 (2018).

Mammosphere Growth 6.

Fettig LM et al. Cross talk between progesterone receptors and retinoic acid receptors in regulation of cytokeratin 5-positive breast cancer cells. Oncogene, 36(44);6074-6084 (2018)

Neurosphere Growth Kinetics 7.

Griesinger AM et al. Neuro NF-κB upregulation through epigenetic silencing of LDOC1 drives tumor biology and specific immunophenotype in Group A ependymoma . Neuro-Oncology, Volume 19, Issue 10, 1 October;1350–1360 (2017)

Live-Cell Analysis Handbook — Second Edition

Kinetic Multi-Spheroid Assays

Key advantages:

Integrated turnkey solution for reproducible analysis of multi-spheroid cultures

Introduction The use of multi-cell tumor spheroids as a model for oncology research has expanded rapidly in recent years. As 3D spheroid protocols become more accessible to researchers, the experimental models have become more complex with correspondingly greater translational potential. Multi-spheroid models have a number of advantages over conventional cell culture. In 2D cell culture, tumor cells are grown in monolayers under conditions that are quite different from the physiological conditions of a tumor. They are grown on a rigid, non-biological surfaces, have an abundance of oxygen, and an excess of nutrients produces hyper-nourished cells with unrestricted and non-physiological proliferation characteristics. Using such cells for drug screening introduces unwanted bias, with a tendency to identify molecules that work only against a uniform population of proliferative cells, and overestimating efficacy. A more realistic approach involves recreating more physiologic heterogeneity inherent to a 3D tumor structure and providing a microenvironment more closely recapitulating in vivo conditions, which includes key interactions between the tumor and the extracellular matrix (ECM). Scaffold-based 3D spheroid models, in which tumor cell aggregates are grown in ECM scaffolds such as Matrigel®, more closely recapitulate physiological growth conditions of tumors, enabling the study of interactions between tumor cells and the microenvironment. Tumor cells grown in 3D scaffold-based culture can form cell-cell and cell-matrix interactions. The heterogeneous nature of the tumor microenvironment can also be studied through the use of co-culture models, such as tumor cells with fibroblasts or immune populations. 1 Coat plate (Day 0)

2 Add cells (Day 0)

This chapter illustrates how IncuCyte’s multi-spheroid assays can provide a rapid method for the pharmacological investigation of potential drug candidates. The approach allows the measurement of real-time viability and toxicity measurements in a meaningful, multicellular in vitro model that better reflects the heterogeneous nature of tumors and allows deeper study of their potential physiological interactions with the tumor microenvironment.

IncuCyte® S3 Multi-Spheroid assays at a glance The IncuCyte® S3 Multi-Spheroid assays combine IncuCyte’s proprietary DF Brightfield image acquisition mode with a 3D multispheroid model grown on a layer of extracellular matrix in a 96well format. Multiple protocols have been developed to suit various experimental objectives and cost considerations. After coating either flat-bottom or round-bottom culture plates1 with Matrigel®, cells are added to the wells with or without IncuCyte® Cell Health Reagents. An optional layer of Matrigel can be added to “sandwich” the cultures and surround them with ECM. The cells are then automatically scanned in the IncuCyte® S3 Live-Cell Analysis System every six hours to monitor multi-spheroid formation. After treatments are added, the spheroid growth and shrinkage assay is initiated and monitored by repeat scanning every six hours in the IncuCyte® S3 system for up to two weeks. Spheroid size is reported based on DF Brightfield image analysis and can be accessed in real time or on demand as needed. The simple protocol is shown below:

3 Add reagent (Day 0, optional)

4 Monitor formation (Day 0—3)

• Quantify label-free growth and investigate morphology of multiple spheroids grown on ECM inside a standard tissue culture incubator, ensuring improved physiological relevance.

• Generate reproducible data suitable for pharmacological analysis with lab-tested and cost-conscious protocols, high quality images, and unbiased analysis.

• Multiplex kinetic viability and toxicity measurements using IncuCyte® non-perturbing Cell Health Reagents to investigate drug mechanisms of action.

• Rapidly create concentration response curves from thousands of images generated in 96-well plates.

Application of IncuCyte® S3 Multi-Spheroid live-cell assays Combining the IncuCyte® DF Brightfield image acquisition with a 3D multi-spheroid cell model on ECM allows for monitoring and quantification of changes over time and under more physiological relevant conditions. As the spheroids remain undisturbed within

the incubator while images are taken, it is possible to visualize and quantify spheroid growth, measure cell health, and better understand mechanisms of drug action.

Label-free image acquisition using novel DF-Brightfield reveals morphology and enables quantification of spheroid growth over time Introduction of a new, enhanced depth of focus brightfield (DF Brightfield) image acquisition enables imaging of multiple tumor spheroids on a layer of extracellular matrix (Figure 2). This enhanced image acquisition results in high contrast brightfield images that can be reliably segmented and analyzed using IncuCyte’s purpose-built processing algorithms. Traditional Brightfield

The novel DF Brightfield image capture provides high-quality images of multi-spheroids formed from a range of tumor cells (Figure 3). Within just three days, different tumor cell lines begin to show distinctive morphology, demonstrating the physiological relevance of the models.

DF Brightfield

Figure 2. IncuCyte’s proprietary image acquisition technique, DF Brightfield for 3D Cultures, generates high contrast, extended depth of focus images when used in conjunction with the IncuCyte S3 Multi-Tumor Spheroid protocol. Subsequent segmentation and analysis with IncuCyte S3’s image processing algorithms results in reproducible data without operator bias.

Lung A549 "Round"

5 Add treatments (Day 3)

Ovarian SKOV-3 "Round"

Breast MCF-7 "Round"

Breast MDA-MB-231 "Stellate"

HD Phase Image

Coat plate (50% Matrigel, 40 µl/well). Polymerize at 37°C for 30 minutes.

Add cells in media (100 or 150 µl/well) with or without cell health reagent, respectively.

Add cell health reagent (50 µl/well) at 3x final assay concentration.

Place inside the IncuCyte and scan every six hours to monitor multi-spheroid formation.

Add treatments and continue to monitor growth in IncuCyte.

DF-BF Image

Figure 1. Overview of workflow for generation and analysis of multi-spheroid cultures in an IncuCyte® S3 Live-Cell Analysis System. 1. Protocols have been developed using either 96-well flat bottom or round bottom plates to address cost concerns when utilizing Matrigel. Round bottom plate protocols use four times less Matrigel than flat bottom plate protocols. 74

Figure 3. High quality HD Phase and DF Brightfield (DF BF) images of multi-spheroids (MS) formed from a range of tumor cell lines (5 d post seeding) on a Matrigel® base. Three day post seeding, A549, SKOV-3 and MCF-7 cells formed round aggregates, while MDA-MB-231 MS exhibited stellate branching distinctive of an invasive morphology. 75

Live-Cell Analysis Handbook — Second Edition IncuCyte S3 multi-spheroid automated image acquisition and analysis software tools enable label-free, kinetic quantification of growing and shrinking multi-spheroids over time. Figure 4 (below) illustrates the growth curves of three multi-spheroid tumor models over several days and demonstrates the inhibitory effects of the cytotoxic drug camptothecin (CMP) on their growth trajectories.

Vehicle

Kinetic Multi-Spheroid Assays Segmented DF Brightfield images compare vehicle or CMP treated conditions at 168 h. Time courses show the individual well total Brightfield Object area (µm2) (y-axis) over 168 h and illustrate specific cell type-dependent kinetic profile of spheroid growth and shrinkage. Data were collected over 168 hour period at 6 hour intervals. All images captured at 10x magnification. Each data point represents mean ± SEM, n=6 wells.

CMP (1µM)

Fluorescent readouts of stably expressing nuclear restricted fluorescent protein act as a surrogate for cell viability When a range of antibodies were tested against different cell surface markers in various lymphocyte cell lines (Figure 4) it was found that the internalization patterns corresponded with the known surface marker expression of these cells. Labeling antibodies with IncuCyte® FabFluor-pH Red showed that anti-CD20 was internalized in a B-cell line, but not a T-cell line, whereas anti-CD3 was internalized in T-cells and not B-cells. Antibodies to CD71 and CD45, which are general lymphocyte markers, were internalized in both cell types. Crucially, the non-specific mouse IgG was not internalized in any of the cell lines. These data demonstrate the broad application and specificity across different cell types and molecules.

A549

MCF-7

MDA-MB-231

Figure 4. Real-time quantification of the growth of A549, MCF-7 and MDA-MB-231 spheroids. Cells were seeded in flat-bottom 96-well plates (2,000 cells per well) on a bed of Matrigel® and spheroids allowed to form (72 h). Spheroids were treated with either vehicle (0.1% DMSO) or CMP (10 µM).

Figure 5. Analysis of spheroids expressing IncuCyte® NucLight Red fluorescent proteins enables determination of spheroid viability. Representative images taken at 144 h show a strong red fluorescent signal in a vehicle control spheroid, in contrast to a marked loss of red fluorescence in the CMP treated spheroid. The yellow boundary in the images represents the DF Brightfield mask outline. Monitoring the integrated intensity from within the DF Brightfield boundary highlights increasing fluorescence under vehicle control conditions corresponding to the growth of the spheroid. Upon treatment with CMP (0.4 nM - 1 µM), a concentration-dependent reduction in integrated fluorescence is observed, with complete loss of fluorescence at the highest concentration tested after 144 h. 76

Live-Cell Analysis Handbook — Second Edition

Multiplexed viability and toxicity measurements enable investigation of mechanisms of action In addition to screening for cytotoxicity, live-cell multi-spheroid assays can be used in conjunction with IncuCyte® Cell Health Reagents to determine the mechanism of action of drugs by combining data on size with levels of cytotoxicity and apoptosis (Figure 6). Here, the power of the assay is illustrated, distinguishing the cytotoxic effects of CMP from that of cycloheximide (CHX). The ability to multiplex reagents with fluorescent-expressing cell lines provides a depth of information on drug mechanism of action under realistic physiological conditions.

By enabling long term, continuous live-cell analysis under uninterrupted incubation, the IncuCyte® S3 multi-spheroid assay makes it possible to derive meaningful EC50 values for drugs by deriving concentration response curves over several days without disturbing cell biology. In the figure below (Figure 7) this is demonstrated for CMP and CHX, and this approach is used to further validate drug mechanism of action observed through earlier analysis.

CMP — Cytotoxic mechanism

CHX — Cytototatic mechanism

Figure 7. Determination of EC50s of CMP and CHX using the IncuCyte S3 Live-cell Analysis System to confirm mechanisms of action in response to cytotoxic (CMP) and cytostatic (CHX) drugs. IncuCyte A549 NucLight Red Cells (2k/well, nuclear-restricted FB indicating viability) were seeded in the present of IncuCyte V Annexin Green reagent (1%, apoptosis marker) and spheroids allowed to form for 3 days. Spheroids were then treated with a concentration range of either CMP, CHX, or vehicle control, and the cells were imaged every 6 h for 7 d. Both CMP (cytotoxic) and CHX (cytostatic) drugs caused a concentrationdependent inhibition of spheroid growth (total BF time courses, not shown). The CMP EC50, using AUC from 0-7 d after treatment, showed a concentrationdependent loss of viability and increase in apoptosis, while CHX EC50s showed little change in viability and no increase in apoptosis.

Reproducible, quantitative data at throughput suitable for pharmacological testing A pharmacological study performed in MCF-7 breast cancer cells illustrates the potential of the IncuCyte’s multi-spheroid assays for drug toxicity testing (Figure 8, below). The ability to view spheroids in 96-well plates over time provides a rapid method for comparing the potencies and toxicities of different compounds within the same assay.

Figure 6. Cytotoxic and cytostatic mechanisms of action can be differentiated by measuring spheroid size and apoptosis. A549 cells were plated at a density of 2,000 cells per well and spheroid allowed to form (96 h). Spheroids were treated with increasing concentrations of CMP (top row, 4 nM – 10 µM) or CHX (bottom row, 4 nM – 10 µM) in the presence of IncuCyte® Annexin V Green reagent (1%). Images were taken every 6 h for 10 d. Time courses show change in size (Brightfield area) or apoptosis (IncuCyte Annexin V fluorescence intensity) over time. CRCs show the different profiles of cytotoxic and cytostatic mechanisms. 78

Figure 8. Effect of CMP, cisplatin (CIS) and oxaliplatinin (OXA) on growth of MCF-7 cells in a 3D spheroid assay. MCF-7 cells were plated at a density of 1,000 cells per well and spheroids allowed to form (72 h). Cells were then treated with serial dilutions of compounds and the kinetics of spheroid growth were obtained. Plate view shows the individual well Total Brightfield area (µm2) over time. Concentration response curves represent the area under curve of the Total Brightfield area time course (µm2) from 0 - 168 h post-treatment. Data were collected over 168 h period at 6 h intervals. Each data point represents mean ± SEM, n=3 wells. 79

Live-Cell Analysis Handbook — Second Edition

Conclusions The use of multi-spheroid assays as model systems for the evaluation of new treatments and study of disease has expanded in recent years. However, despite their ability to measure indicators of cell health, such as cytotoxicity and apoptosis, common assays rely on indirect, end-point measurements that are subject to artefacts and cannot be readily verified by morphological changes. The IncuCyte S3 Live-Cell Analysis System’s Multi-Spheroid assay is a rapid method for the pharmacological investigation of potential drugs in a meaningful multicellular model, which allows researchers to:

•• Link morphology, growth, and toxicity via direct, kinetic measurements by combining novel DF Brightfield image acquisition and analysis with IncuCyte® Cell Health Reagents.

•• Study spheroid morphology, growth and shrinkage using a label-free, scaffold-based, kinetic 96-well plate assay without removing cultures from the incubator and without need for a predefined endpoint selection.

•• Carry out automated analysis of multiple conditions or variables under more physiologically relevant conditions, minimizing interference with cell biology.

•• Investigate mechanisms of action with multiplexed viability and toxicity measurements using non-perturbing reagents and cell lines that stably express fluorescent proteins. •• Produce reproducible well-to-well kinetic data in live spheroid cultures, in response to different compounds and conditions, using lab-tested protocols, high quality images, and unbiased analysis.

Kinetic Single Spheroid Assays Reproducible quantitative analysis of single spheroid growth and health over time

Introduction Single spheroids exhibit several physiological traits including relevant morphology, increased cell survival, and a hypoxic core that make them ideally suited to the study of larger solid tumors. These scaffold-free (media-based) models are easily achieved using round-bottom ultra-low attachment (ULA) microplates to promote spheroid self-assembly. They generally yield a single tumor spheroid per well. By virtue of their size, they exhibit the key features of solid tumors: they are comprised of proliferating and quiescent cells, and contain necrotic zones resulting from a gradient of nutrients, metabolites and oxygen that recapitulates the authentic heterogeneity within a tumor. Single spheroids formed in ultra-low attachment (ULA) plates also avoid the use of complex and poorly characterized biomatrices, and are ideal for studies that require high levels of well-to-well consistency. A growing body of evidence suggests that more relevant and translational observations can be made in 3D multi-cell tumor models compared to 2D monolayer models. Most currently 1 Cell seeding (Day 0)

available 3D techniques for generating and quantifying spheroids are time consuming, laborious, costly and can lack reproducibility. In this chapter we describe the validation and application of miniaturized IncuCyte live-cell single tumor spheroids formed in ULA plates and monitored up to two weeks. These assays are flexible, simple to run, and provide automated and direct measures of tumor size and health in real time.

IncuCyte® S3 Single Spheroid assays at a glance Cells of interest are harvested, counted, and plated onto ULA round bottom 96- or 384-well plates and then centrifuged. Spheroid formation is monitored every six hours using DF Brightfield and HD phase contrast image acquisition until they reach the desired size. Test compounds are then added, and the spheroid growth and shrinkage assay is initiated. Automated monitoring continues every six hours for up to two weeks, with tumor spheroid size measurements made available in real time. The simple protocol is shown below:

2 Spheroid formation (Day 0—3)

Seed cells into 96W or 384W Ultra Low Attachment plate. Centrifuge.

Place inside the IncuCyte® S3 and scan every six hours.

3 Add treatments (Day 3)

Add treatments to plate. Monitor spheroid growth and shrinkage.

Figure 1. Overview of workflow for generation and analysis of single spheroid cultures in an IncuCyte® S3 Live-Cell Analysis System.

Key advantages: • Quantify growth and investigate morphology of single spheroids grown in ultra-low attachment (ULA) round bottom plates without the need of labels, and leaving cells undisturbed inside a standard tissue culture incubator. • Investigate mechanisms of action or immune modulation with kinetic viability and toxicity measurements using nonperturbing reagents.

• Lab-tested protocols, high quality images, and unbiased analysis delivers robust data suitable for pharmacological analysis. • Automated acquisition and analysis tools enable rapid creation of concentration response curves from thousands of images generated from 96- or 384-well plates.

Live-Cell Analysis Handbook — Second Edition

Kinetic Single Spheroid Assays

Application of IncuCyte® S3 Single Spheroid live-cell assays IncuCyte® S3 DF Brightfield Image Acquisition (described in IncuCyte S3 Multi-Spheroid Assays, Figure 2) enables robust measurement of spheroid size, yielding information on spheroid growth rates and morphology without the need for labels. Following treatment with cytotoxic compounds, a strong

Brightfield signature is obtained which, when combined with fluorescence analysis using IncuCyte® Cell Health Reagents, provides insight into the viability of tumor spheroids in response to different treatments.

Label-free monitoring of spheroid growth and morphology over time The example below illustrates how the IncuCyte S3 DF Brightfield analysis can provide kinetic measurements of single spheroid size over time (Figure 2). The size of tumor spheroids was measured using an automated algorithm that masked the largest Brightfield

Breast MDA-MB-231 + Matrigel

Reproducible, quantitative data to conduct pharmacological analysis To demonstrate the pharmacological utility of the single spheroid assay, we performed a comparison of three different drugs using 3D spheroids derived from the SKOV-3 ovarian cancer line (Figure 3). The data shows a concentration-dependent inhibitory growth effect for all compounds, and illustrates how compound potencies can be directly compared within the same assay.

object in the field of view. Changes in the size of spheroids derived from three types of tumor cell were monitored in response to camptothecin, allowing growth response curves to be generated in the absence and presence of the drug.

Connective Tissues HT-1080

CMP

CIS

OXA

Vehicle

Lung A549

Vehicle

CMP (1µM) µM

Figure 2. Brightfield analysis enables accurate kinetic quantification of spheroids. The differential pharmacological effect of 1 µM camptothecin (CMP) on growth of MDA-MB-231, HT-1080 and A549 cells in a 3D spheroid assay. Cells were grown in ULA round-bottom 96- well plates (2,500 cells per well) for 72 h and treatment with ± 1 µM CMP followed. Segmented DF Brightfield images compare treated vs. un-treated conditions at 240 h. Time courses illustrate the specific cell type-dependent kinetic profile of spheroid growth and shrinkage. The graphs display the Largest Brightfield Object area (µm2) (y-axis) over the course of a 240 h assay (x-axis) at 6 h intervals. All images captured at 10x magnification. Each data point represents mean ±SEM, n=4. 82

Figure 3. Effect of CMP, cisplatin (CIS) and oxaliplatin (OXA) on growth of SKOV-3 cells in a 3D spheroid assay. SKOV-3 cells were plated at a density of 5,000 cells per well and spheroid allowed to form (72 h). Cells were then treated with serial compound dilutions and kinetics of spheroid growth and shrinkage were obtained. Plate graph shows the individual well Largest Brightfield area (µm2) over time. IC50 curves represent the Largest Brightfield area (µm2) at 204 h post-treatment. Data were collected over 240 h period at 6 h intervals. Each data point represents mean ±SEM, n=8. 83

Live-Cell Analysis Handbook — Second Edition

Kinetic Single Spheroid Assays

Miniaturizing the 3D spheroid cultures makes it possible to optimize the assay further by testing different seeding densities for multiple cell types in a single experiment. This is demonstrated below using three cell types plated at four different densities (Figure 4). The data demonstrates the cell area dependence with seeding density and differential growth profile across the cell types.

A549

Viability and toxicity measurements using non-perturbing reagents enable investigation of drug mechanisms of action Masking of the DF Brightfield channel allows measurement of other parameters of interest when non-perturbing reagents are employed. In Figure 5 (below) the effects of CMP on cell viability were studied by stably expressing a red fluorescent protein (using IncuCyte NucLight Red Lentiviral Reagent) in the spheroids.

HT-1080

SKOV-3

Vehicle

Applying the ‘fluorescence within the Brightfield boundary’ feature in the IncuCyte® S3 spheroid software module negates the need to apply a fluorescence mask, and removes the impact of threshold settings.

1 µM CMP

Figure 5. Analysis of spheroids expressing fluorescent proteins enables spheroid viability determination. Representative images taken at 240 h show a strong red fluorescent signal in a vehicle control spheroid, in contrast to a marked loss in red fluorescence in the CMP-treated spheroid. The yellow boundary in the images represents the Brightfield mask outline. Monitoring the integrated intensity from within the Brightfield boundary highlights a gradual increase in fluorescence under vehicle control conditions (red symbols) corresponding to the growth of the spheroid. Upon treatment with CMP, a concentration-dependent reduction in integrated fluorescence is observed, with abolishment of fluorescence with the highest concentration tested after 240 h.

Cell density A similar approach was used with IncuCyte® Cell Health Reagents to determine the mechanism of cell death after treatment with CMP (Figure 6). Little or no reduction in spheroid size was observed with the Brightfield phase contrast images, but fluorescence

Vehicle

Figure 4. Miniaturizing spheroid growth and shrinkage assay for assay optimization. Comparison of temporal growth profiles of A549, HT-1080 and SKOV-3 cells in a miniaturized 3D spheroid assay. All cells seeded at a density ranging from 310 to 7,500 cells per well plated in a ULA round-bottom 384-well plate. Media was replenished 72 h post seeding. Microplate overview image shows DF Brightfield segmentation mask at 204 h post-media replenishment. Histogram compares the distribution frequency of the Brightfield area (µm2) across all cell types plated at 2,500 cells per well at this time point. Variability plot analysis shows the largest Brightfield area of individual wells at 204 h. Time-course plots represent the differential temporal profile of the Largest Brightfield Object Area metric (µm2) across the cell types. Data were collected over a 204 h period at 6 h intervals, all images captured at 10x magnification. Each data point represents mean ±SEM, n=32. 84

analysis revealed a concentration-dependent effect with increases in the mean intensity within the Brightfield boundary, suggesting induction of apoptosis.

1 µM CMP

Figure 6. Effect of CMP on A549 cells reported by IncuCyte® Annexin V Green reagents in a 3D spheroid assay. A549 cells were seeded at a density of 2,500 cells per well in ULA round bottom plates and spheroids were formed for 96h. Spheroids were treated with CMP (0.4 nM - 1 µM) or vehicle (0.1% DMSO) and apoptosis was reported using IncuCyte Annexin V Green. 85

Live-Cell Analysis Handbook — Second Edition Finally, data is presented showing how combining the Brightfield and Cell Health readouts can be used in single spheroids to determine the mechanism of action of compounds (Figure 7). CMP caused a marked increase in fluorescence intensity, suggesting a cytotoxic mechanism, whereas CHX only caused a notable increase in fluorescence at the highest concentration tested. The clear separation between the size and cytotoxicity readouts supports the known cytostatic mechanism of CHX.

Kinetic Single Spheroid Assays

Flexible assay format can be adapted for co-culture studies using relevant immune-oncology cell models In order to further demonstrate the utility of the IncuCyte® S3 Single Spheroid Assay as a translational cell-based assay, an immune cell killing model was optimized and employed to assess the efficacy of novel immune modulators. As illustrated below, new insights for greater translational relevance can be

gained by utilizing 3D ADCC spheroid assays, as compared to 2D ADCC assays, for the examination of immune modulators, such as Herceptin. Such models may uncover important information regarding dose response kinetics.

Figure 8. Herceptin-induced ADCC in HER2-positive SKOV-3 cells. HER2-positive SKOV-3 or HER2 negative A549 NucLight Red™ spheroids (2.5K/ well) were seeded with PBMCs (6.25K/well) and treated with Herceptin (mAb targeting HER2 receptors). Herceptin-induced cytotoxicity was observed and measured in SKOV-3 but not A549 spheroids. A similar assay was conducted in a 2D culture model. SKOV-3 cells (1.6K/well) were seeded overnight prior to the addition of PBMCs (8K/well) and subsequent treatment with Herceptin. SKOV-3 tumour spheroids appear to exhibit ~300-fold lower Herceptin sensitivity in comparison to 2D. Note the apparent 34% inhibition of the 3D spheroid at the lowest test concentration (0.08 µg/ml), suggesting that a biphasic concentration response curve may exist; the outermost cells behave as in the 2D model, whereas the spheroid center has lower sensitivity. Figure 7. Cytotoxic and cytostatic mechanisms of action can be differentiated by measuring spheroid size and viability. SKOV-3 cells were plated at a density of 2,500 cells per well and spheroid allowed to form (96 h). Spheroids were treated with increasing concentrations CMP (0.5 nM – 1 µM) or CHX (1.4 nM – 10 µM) in the presence of IncuCyte® Cytotox Green reagent (25 nM). Images were taken every 6h for 10 days. Time courses show change in Brightfield area (top row) or fluorescent response (bottom row) over time. CRCs show the different profiles of cytotoxic and cytostatic mechanisms.

Live-Cell Analysis Handbook — Second Edition

Conclusions 3D micro-tissues and organoids are becoming increasingly popular for their more relevant morphology and cell survival, but current methods for assessing the growth and vitality of these models are limited. Most assays require cell labeling and only provide a single time point readout. By contrast, the IncuCyte® S3 Live-Cell Assay allows:

•• Miniaturization of the assay to provide a high-throughput method of producing reproducible, quantitative pharmacological data in physiologically relevant cell models simultaneously for a variety of test compound.

•• Label-free study of spheroid morphology, growth and shrinkage in 96- and 384-well format.

•• IncuCyte® S3 Live-Cell Single Spheroid Assay provides a valuable and timely addition to the toolkit of those working in basic biology, medical research, and pharmacological drug development who seek to better understand cellular responses.

•• Generation of kinetic measurements and cell-dependent growth curves for spheroids derived from different types of cells. •• Monitoring of spheroid viability over time and in different treatment conditions using IncuCyte’s Cell Health Reagents and/ or stable expression of fluorescent reporter proteins and the ability to link this to morphological observations.

•• Flexibility in cell types and co-culture, enabling extension of the assay for study of immune modulators.

Appendix

Live-Cell Analysis Handbook â&#x20AC;&#x201D; Second Edition

IncuCyte® Cell Proliferation Assay Protocol For label-free proliferation measurements of adherent or non-adherent cell lines

This protocol provides an overview of the IncuCyte® Cell Proliferation Assay methodology. It is compatible with the IncuCyte® live-cell analysis system using your choice of cells and treatments. The highly flexible assay format can be combined with our range of IncuCyte® cell health and viability reagents for multiplexed measurements of cytotoxicity and apoptosis alongside proliferation in the same well.

Required materials • Flat bottom tissue culture plate (e.g., Corning 3595) • Poly-L-ornithine (Sigma P4957) – optional, for non-adherent cells • Fibronectin (Sigma F1141) – optional, for non-adherent cells

General guidelines •• Following cell seeding, place plates at ambient temperature (15 minutes for adherent cell lines and 45 minutes for non-adherent cell lines) to ensure homogenous cell settling. •• Remove bubbles from all wells by gently squeezing a wash bottle containing 70-100% ethanol with the inner straw removed, to blow vapor over the surface of each well. •• After placing the plate in the IncuCyte live-cell analysis system, allow the plate to warm to 37°C for 30 minutes prior to scanning.

Adherent cell line protocol

Coat wells

Coat wells of plate (50 μL/well) with appropriate matrix. Optional for adherent cell lines.

Plate cells

Seed cells (100 μL/well, 1,000–10,000 for adherent and 5,000–50,000 for non-adherent) into a 96-well plate.

Add treatments

Add desired treatments (100 μL/well, 1x for adherent cell lines, 2x for non-adherent cell lines).

Live-Cell Analysis Handbook — Second Edition

IncuCyte® Cell Proliferation Assay Protocol

DAY 0: 1

Coat wells (optional)

Add treatments 3.1. Immediately after cell seeding, add treatments and controls to appropriate wells of the 96-well plate containing cells. Triturate wells to appropriately mix the treatment to ensure cell exposure at 1x.

Plate cells

3.2. Place the cell plate into the IncuCyte live-cell analysis system and allow the plate to warm to 37°C for 30 minutes prior to scanning.

2.1. Seed your choice of cells (100 μL per well) at an appropriate density into a 96-well plate, such that by day 1 the cell confluence is approximately 10-20%. The seeding density will need to be optimized for the cell line used; however, we have found that 1,000 to 2,500 cells per well (10,000 to 25,000 cells/mL seeding stock) are reasonable starting points. a. Monitor cell growth using the IncuCyte® system to capture phase contrast images every 2 hours and analyze using the integrated confluence algorithm. 3

1.1. Depending on cell line used, coat a 96-well flat bottom plate with relevant coating matrix according to manufacturer’s recommendation.

a. Objective: 4x (recommended 1 image per well or whole well) or 10x b. Channel selection: Phase Contrast (+ “Green” or “Red” if fluorescent label or cell health reagents are used) c. Scan type: Standard d. Scan interval: Typically, every 1 to 2 hours, until your experiment is complete.

Add treatments

3.1. Prepare 1x concentrations of desired cell treatments in cell culture medium. The volumes may be varied; however, we recommend preparing enough volume of each desired treatment/dilution in order to achieve 100 μL per well. 3.2. Remove the cell plate from the incubator and aspirate medium from wells. 3.3. Add treatments and controls to appropriate wells of the 96-well plate. 3.4. Place the cell plate into the IncuCyte live-cell analysis system and allow the plate to warm to 37°C for 30 minutes prior to scanning. a. Objective: 4x, 10x or 20x b. Channel selection: Phase Contrast (+ “Green” or “Red” if fluorescent label or cell health reagents are used) c. Scan type: Standard d. Scan interval: Typically, every 1 to 2 hours, until your experiment is complete

Non-adherent cell line protocol This protocol can also be used to evaluate immune cell clustering and proliferation following activation. DAY 1: 1

Seed cells and add prepared treatments 1.1. Coat a 96-well flat bottom plate with relevant coating matrix. We recommend coating with 50 μL of either 0.01% polyL-ornithine solution or 5 μg/mL fibronectin diluted in 0.1% BSA. Coat plates for 1 hour at ambient temperature, remove solution from wells, then allow plates to dry for 30-60 minutes prior to cell addition. 1.2. Prior to cell seeding, prepare cell treatments at 2x final assay concentration in enough cell culture medium to achieve a volume of 100 μL per well.

Plate cells 2.1. Seed your choice of cells (100 μL per well) at an appropriate density into a 96-well plate. The seeding density will need to be optimized for the cell line used; however, we have found that 5,000 to 50,000 cells per well (50,000–500,000 cells/mL seeding stock) are reasonable starting points. NOTE: If studying immune cell clustering and proliferation, prepare cell activation treatments at 5x final concentration, and immediately add 50 μL per well containing cells. It is advised that some control wells containing only vehicle are included in the plate.

Live-Cell Analysis Handbook — Second Edition

IncuCyte® Cell Transduction Protocol

Live cell fluorescent imaging

For stable or transient fluorescent labelling of cell nuclei

4.1. Place the plate into the IncuCyte system and allow theplate to warm to 37°C for 30 minutes prior to scanning. a. Objective: 4x, 10x or 20x b. Channel selection: Phase + Fluorescence c. Scan type: Standard d. Scan interval: Typically every 2 hours.

Nuclear labeling by transient transduction

This protocol provides an overview of methodologies used for the transduction of cell lines with a range of IncuCyte® NucLight labeling reagents. The IncuCyte NucLight live-cell labelling reagents are used to fluorescently label the nuclei of living cells without perturbing cell function or biology, enabling real-time cell counting using your choice of cells and treatments. Transient or stable expression strategies are supported using either the IncuCyte® NucLight BacMam 3.0 or NucLight Lentivirus reagents, respectively. In addition, the highly flexible assay format can be combined with our range of IncuCyte® Cytotox Reagents or the IncuCyte® Caspase-3/7 reagent for multiplexed measurements of cytotoxicity and apoptosis alongside proliferation in the same well. 1

Harvest cells

Transduce

DAY 1:

Addition of treatment

We recommend the use of IncuCyte® NucLight BacMam 3.0 Reagents to transiently transduce your choice of cells. As well as eliminating the need to create stable cell lines these reagents enable the rapid expression of a nuclear-restricted green or red fluorescent protein (GFP or mKate2) in your choice of primary, immortalized, dividing or non-dividing mammalian cells without altering cell function and with minimal toxicity.

1.1. Once the infected cells have started to fluoresce (usually after 18 - 21 hours), remove the cell plate from the incubator and add desired treatments. The volumes/dilutions may be varied; however, we recommended aspirating the culture medium and adding 100 μL of the desired treatment, prepared at 1x final assay concentration. Continue to image the plate in the IncuCyte system until your experiment is complete.

Creating a cell population or clone that stably expresses a nuclear label 3

Seed cells

We recommend the use of IncuCyte NucLight Lentivirus Reagents to provide stable, homogenous expression of a nuclear-restricted green or red fluorescent protein (GFP or mKate2) in your choice of living mammalian cells without perturbing cell function and with minimal toxicity. These reagents are ideal for generating stable cell populations or clones using puromycin or bleomycin selection.

Live cell fluorescent imaging

Protocol Seed cells

1 Harvest cells and resuspend at 2 x 104 cells/mL in full growth medium.

Add Green or Red NucLight BacMam 3.0 Reagent (1 to 2% (v/v)). Mix by inversion.

Seed cells (100μL/well, 2 x 103 cells/ well) and incubate at ambient temperature for 30 minutes.

Seed cells in growth media and leave to adhere (4-24 hours). Cells should be 1535% confluent at the time of transduction.

DAY 0:

Harvest cells 1.1. Harvest cells and dilute to 20,000 cells/mL in a conical tube. Note: Cultures should be below 80% confluence at time of harvest, and at least 90% viable, prior to transduction.Overly confluent or unhealthy cells will result in inefficient transduction.

Apply selection

Live cell fluorescent imaging

Add Green or Red NucLight Lentivirus Reagent (MOI 3 to 6) diluted in media ± Polybrene®. After 24 hours, replace the media with fresh growth media. Monitor expression using the IncuCyte system.

Apply anitobiotic selection to derive a stable, homogenous cell population or clone that expresses a nuclear restricted green or red fluorescent protein. Optional: Freeze cells and use for future assays.

Capture images every 1 to 2 hours (4x, 10x or 20x) in an IncuCyte system. Analyze using integrated software.

DAY 0: 1

Transduce

Seed cells 1.1. Seed cells in growth media of choice and at a density such that they are 15-35% confluent after 24 hours of incubation.

2.1. Add IncuCyte NucLight BacMam 3.0 reagent to cells and mix by inversion. The concentration of IncuCyte NucLight BacMam 3.0 reagent used will need to be optimized for each cell type used; however we have found that concentrations of 0.5 to 4% (v/v) are reasonable starting points. We recommend running our optimization protocol prior to setting up proliferation assays. 3

Transduce

Capture images every 1 to 2 hours (4x, 10x or 20x) in an IncuCyte system. Analyze using integrated software.

Protocol 1

Seed cells 3.1. Seed the transduced cells (100 μL per well, 2,000 cells per well) into a 96-well flat bottom plate and allow the plate to incubateat ambient temperature for 20 minutes prior to scanning.

Live-Cell Analysis Handbook — Second Edition

Transduce 2.1. Add the IncuCyte NucLight Lentivirus Reagent at desired multiplicity of infection (MOI = TU/cell). An MOI of 3 to 6 is recommended for most cell types, however, an optimized MOI should be determined for each cell type used. Polybrene® (1-8 μg/mL) may also be added to enhance transduction of some cell types (Note: Certain cell types can be sensitive to Polybrene® (e.g. neurons)). 2.2. Incubate at 37°C, 5% CO2 for 24 hours, then remove and replace with fresh growth media. 2.3. Return to incubator for an additional 24-48 hours, monitoring expression using an IncuCyte system

Apply selection 3.1. Remove media and replace with fresh growth medium containing selection (i.e. puromycin or bleomycin). Example: For HT-1080, A549, HeLa, and MDA-MB-231 cells, complete media containing 1 μg/mL puromycin is sufficient for efficient killing of non-transduced cells.

Live cell fluorescent imaging 4.1. Incubate for 72-96 hours, replacing media every 48 hours. Maintain stable population in a maintenance concentration of selection media. Example: HT-1080, A549, HeLa, and MDA-MB-231 cells labeled with the IncuCyte NucLight Red Lentivirus Reagent (EF-1 μ, Puro) can be maintained in complete media containing 0.5 μg/mL puromycin.

IncuCyte® Apoptosis Assay Protocol For the fluorescent detection of caspase-3/7 activation or phosphatidylserine externalization

This protocol provides an overview of the IncuCyte Apoptosis Assay methodology which uses mix-and read IncuCyte® Caspase 3/7 or Annexin V Reagents to detect apoptosis in real time. It is compatible with the IncuCyte® live-cell analysis system using your choice of cells and treatments. The highly flexible assay format can be combined with our range of IncuCyte® NucLight red nuclear labeling reagents or labeled cell lines for multiplexed measurements of proliferation and apoptosis in the same well.

Required materials • IncuCyte® Caspase- 3/7 Apoptosis Reagent (EssenBioscience Cat #4440) or • IncuCyte® Annexin V Red Reagent (EssenBioScience Cat #4641) or

• IncuCyte® Anexin V Green Reagent (EssenBioScience Cat #4642) • Poly-L-ornithine (Sigma P4957) – optional, for non-adherent cells • Fibronectin (Sigma F1141) - optional, for non-adherent cells • Flat bottom tissue culture plate (e.g., Corning 3595)

•• Following cell seeding, place plates at ambient temperature (15 minutes for adherent cell lines and 45 minutes for nonadherent cell lines) to ensure homogenous cell settling. •• Remove bubbles from all wells by gently squeezing a wash bottle (containing 70-100% ethanol with the inner straw removed) to blow vapor over the surface of each well. •• After placing the plate in the IncuCyte® live-cell analysis system, allow the plate to warm to 37°C for 30 minutes prior to scanning. •• If monitoring apoptosis in primary neuronal cultures, we recommend use of the IncuCyte Annexin V Red reagent to eliminate risk of green channel excitation issues in these sensitive cell lines.

Live-Cell Analysis Handbook — Second Edition

IncuCyte® Apoptosis Assay Protocol

Non-adherent cell line protocol

Adherent cell line protocol 1

Seed Cell

Seed cells (100 μL/well, 1,0005,000) into a 96-well plate and incubate overnight.

Prepare apoptosis reagent and treat cells

Prepare the desired treatments at 1x in medium containing IncuCyte Caspase-3/7 or Annexin V Reagents. Aspirate media from wells and add treatment (100 μL/well).

Live cell fluorescent imaging

Capture images every 2-3 hours (20x or 10x) in the IncuCyte® System. Analyze using integrated software.

Coat plate

Prepare incucyte apoptosis reagent and treatment

Coat plate with 0.01% polyL-ornithine solution or 5 μL/mL fibronectin diluted in 0.1% BSA.

Dilute apoptosis reagent in medium and prepare cell treatments

DAY 0: 1

Seed cell 1.1. Seed your choice of cells (100 μL per well) at an appropriate density into a 96-well plate, such that by day 1 the cell confluence is approximately 30%. The seeding density will need to be optimized for the cell line used; however, we have found that 1,000 to 5,000 cells per well (10,000–50,000 cells/mL seeding stock) are reasonable starting points.

Seed cells and add treatment

Seed cells (100 μL/well, 5,000–25,000 cells) into the coated 96-well plate. Immediately add apoptosis reagent ± treatments and triturate.

Live cell fluorescent analysis

Capture images every 2-3 hours (20x or 10x) in the IncuCyte® system.

DAY 1: 1

Coat plate 1.1. Coat a 96-well flat bottom plate with appropriate coating matrix. We recommend coating with 50 μL of either 0.01% poly-L-ornithine solution or 5 μg/mL fibronectin diluted in 0.1% BSA. Coat plates for 1 hour at ambient temperature, remove solution from wells, then allow plates to dry for 30-60 minutes prior to cell addition.

a. Monitor cell growth using the IncuCyte system to capture phase contrast images every 2 hours and analyze using the integrated confluence algorithm. 2

DAY 1: 2

Prepare apoptosis reagent and treatments 2.1. Prior to cell seeding, dilute apoptosis reagents in desired medium formulation.

Apoptosis reagent preparation and cell treatment addition

a. If using caspase-3/7, dilute reagent to a final concentration of 5 μM (1:1000 dilution).

2.1. Dilute apoptosis reagents in desired medium formulations

b. If using Annexin V reagents, solubilize Annexin V by adding 100 μL of complete medium or PBS. The reagents may then be diluted in complete medium containing at least 1 mM CaCl2 for a final dilution of 1:200.

a. If using caspase-3/7, dilute reagent to a final concentration of 5 μM (1:1000 dilution) b. If using Annexin V reagents, solubilize Annexin V by adding 100 μL of complete medium or PBS. The reagents may then be diluted in complete medium containing at least 1 mM CaCl2 for a final dilution of 1:200.

NOTE: All test agents will be diluted in this reagent-containing medium, so make up a volume that will accommodate all treatment conditions. The volumes/dilutions added to cells may be varied; however, a volume of 200 μL per well is generally sufficient for the duration of the assay.

NOTE: All test agents will be diluted in this reagent-containing medium, so make up a volume that will accommodate all treatment conditions. The volumes/ dilutions added to cells may be varied; however, a volume of 100 μL per well is generally sufficient for the duration of the assay.

2.2. Prepare cell treatments at 2x final assay concentration in enough cell culture medium containing caspase-3/7 or Annexin V to achieve a volume of 100 μL per well.

2.2. Remove the cell plate from the incubator and aspirate off growth medium. 2.3. Add treatments and controls to appropriate wells of the96-well plate. 3

3.1. Seed your choice of cells (100 μL per well) at an appropriate density into a 96-well plate in medium containing Caspase 3/7 or Annexin V. The seeding density will need to be optimized for the cell line used; however, we have found that 5,000 to 25,000 cells per well (50,000–250,000 cells/mL seeding stock) are reasonable starting points.

Live-Cell imaging of apoptosis 3.1. Place the cell plate into the IncuCyte Live-Cell Analysis System and allow the plate to warm to 37°C for 30 minutes prior to scanning.

3.2. Immediately add treatments and controls to appropriate wells of the 96-well plate containing cells. Triturate wells to appropriately mix the treatment to ensure cell exposure at 1x.

a. Objective: 10x or 20x b. Channel selection: Phase Contrast and Green (+ “Red” if using fluorescent label or an additional cell health reagent) c. Scan type: Standard (2-4 images per well) d. Scan interval: Typically, every 2 hours, until your experiment is complete.

Seed cells and add prepared treatments

Live-Cell imaging of apoptosis 4.1. Place the cell plate into the IncuCyte® live-cell analysis system and allow the plate to warm to 37°C for 30 minutes prior to scanning. a. Objective: 10x or 20x b. Channel selection: Phase Contrast and Green (+ “Red” if using fluorescent label or an additional cell health reagent) c. Scan type: Standard (2-4 images per well) d. Scan interval: Typically, every 2 hours, until your experiment is complete.

Live-Cell Analysis Handbook — Second Edition

IncuCyte® Cytotoxicity Assay

DAY 0: 1

For the fluorescent quantification of cell death

Seed effector cells

1.1. Seed your choice of cells (100 μL per well) at an appropriate density into a 96-well plate, such that by day 1 the cell confluence is approximately 30%. The seeding density will need to be optimized for the cell line used; however, we have found that 1,000 to 5,000 cells per well (10,000–50,000 cells/mL seeding stock) are reasonable starting points. NOTE: For non-proliferating cell lines (e.g., rate forebrain neurons) we recommend seeding at 15 x 103 to 30 x 103 cells per well, and culturing for 14 days for the neural network to establish, prior to evaluating cytotoxicity. a. Monitor cell growth using the IncuCyte system to capture phase contrast images every 2 hours and analyze using the integrated confluence algorithm.

This protocol provides an overview of the IncuCyte® Cytotoxicity Assay methodology which uses the mix-and-read IncuCyte® Green or Red Reagent to detect cell death in real time. The protocol is compatible with the IncuCyte® live-cell analysis system and your choice of cells (e.g., tumor, immune, neuronal) and treatments. Furthermore, this protocol can be used with cells labeled using the IncuCyte® NucLight nuclear labeling reagents to provide multiplexed measurements of proliferation alongside cell death in the same well.

DAY 1: 2

Required materials

•• We recommend medium with low levels of riboflavin to reduce the green fluorescence background. EBM, F12-K, and Eagles MEM have low riboflavin (<0.2 mg/L). DMEM and RPMI have high riboflavin (>0.2 mg/L).

2.1. Dilute cytotoxicity reagent in desired medium formulation.

• IncuCyte® Red Cytotoxicity Reagent (EssenBioscience Cat #4632)

NOTE: All test agents will be diluted in this reagent-containing medium, so make up a volume that will accommodate all treatment conditions. The volumes/dilutions added to cells may be varied; however, a volume of 100 μL per well is generally sufficient for the duration of the assay.

or • IncuCyte® Green Cytotoxicity Reagent (EssenBioscience Cat #4633)

2.2. Remove the cell plate from the incubator and aspirate off growth medium. 2.3. Add treatments and controls to appropriate wells of the 96-well plate.

• Poly-L-ornithine (Sigma P4957) – optional, for non-adherent cells • Fibronectin (Sigma F1141) - optional, for non-adherent cells

General guidelines

• Flat bottom tissue culture plate (e.g., Corning 3595, TPP 92096 for neuronal cell health)

Cytotoxicity reagent preparation and cell treatment addition

Live-cell imaging of cytotoxicity 3.1. Place the cell plate into the IncuCyte Live-Cell Analysis System and allow the plate to warm to 37°C for 30 minutes prior to scanning. a. Objective: 10x or 20x b. Channel selection: Phase Contrast and Fluorescence

c. Scan type: Standard (2-4 images per well) d. Scan interval: Typically, every 2 hours, until your experiment is complete. NOTE: For neuronal cultures we recommend scanning every 6 to 12 hours to minimize risk of phototoxicity.

•• After placing the plate in the IncuCyte® live-cell analysis system, allow the plate to warm to 37°C for 30 minutes prior to scanning •• If monitoring cytotoxcity in primary neuronal cultures, we recommend use of the IncuCyte® Cytotox Red reagent to eliminate risk of green channel excitation issues in these sensitive cell types.

Adherent cell line protocol 1

Seed cells

Seed cells (100 μL/well, 1,000— 5,000) into a 96-well plate and incubate overnight.

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Prepare cytotoxicity reagent and treat cells

Prepare the desired treatments at 1x in medium containing IncuCyte Cytotoxicity reagent. Aspirate media from wells and add treatment (100 μL/well).

Live cell fluorescent analysis

Capture images every 2-3 hours (20x or 10x) in the IncuCyte® System. Analyze using integrated software.

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Non-adherent cell line protocol 1

Coat Plate

Coat plate with 0.01% poly- L-ornithine solution or 5 μL/mL fibronectin diluted in 0.1% BSA.

Prepare cytotoxicity reagent and treatment

Dilute cytotoxicity reagent in medium at 1x and prepare cell treatments

Seed cells and add treatment

Seed cells (100 μL/well, 5,000– 25,000 cells) into the coated 96-well plate. Immediately add cytotoxicity reagent ± treatments and triturate.

Live cell fluorescent analysis

Capture images every 2-3 hours (20x or 10x) in the IncuCyte® System.

DAY 1: 1

Coat plate

1.1. Coat a 96-well flat bottom plate with appropriate coating matrix. We recommend coating with 50 μL of either 0.01% poly-L-ornithine solution or 5 μg/mL fibronectin diluted in 0.1% BSA. Coat plates for 1 hour at ambient temperature, remove solution from wells, then allow plates to dry for 30-60 minutes prior to cell addition. 2

Prepare cytotoxity reagent and treatments

2.1.Prior to cell seeding, dilute cytotoxicity reagent to a final concentration of 250 nM (1:4000 dilution) in desired medium formulation. NOTE: All test agents will be diluted in this reagent-containing medium, so make up a volume that will accommodate all treatment conditions. The volumes/ dilutions added to cells may be varied; however, a volume of 100 μL per well is generally sufficient for the duration of the assay. 2.2. Prepare cell treatments at 2x final assay concentration in enough cell culture medium the cytotoxicity reagent to achieve a volume of 100 μL per well. 3

Seed cells and add prepared treatments

3.1. Seed your choice of cells (100 μL per well) at an appropriate density into a 96-well plate in medium containing the cytotoxicity reagent. The seeding density will need to be optimized for the cell line used; however, we have found that 5,000 to 25,000 cells per well (50,000–250,000 cells/mL seeding stock) are reasonable starting points. 3.2. Immediately add treatments and controls to appropriate wells of the 96-well plate containing cells. Triturate wells to appropriately mix the treatment to ensure cell exposure at 1x. 4

Live-Cell Imaging of cytotoxicity

1.1. Place the cell plate into the IncuCyte® live-cell analysis system and allow the plate to warm to 37°C for 30 minutes prior to scanning. a. Objective: 4x (recommended 1 image per well or whole well) or 10x b. Channel selection: Phase Contrast and Fluorescence c. Scan type: Standard d. Scan interval: Typically, every 2 hours, until your experiment is complete.

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IncuCyte® Immune Cell Killing Assay For measurements of tumor cell death

This protocol provides an overview for the measurement of immune cell killing of adherent or non-adherent target tumor cells. The flexible assay format is suitable for cytotoxic T cell killing and antibody-dependent cell-mediated cytotoxicity (ADCC) assays using a co-culture methodology that combines direct measurements of tumor cell death with no-wash, mix-and-read protocols. This method utilizes either our IncuCyte® Caspase 3/7 reagent, a substrate that is cleaved during target cell apoptosis to release a green-fluorescent DNA dye that stains the nuclear DNA, or IncuCyte® Annexin V reagent which labels externalized phosphatidylserine (PS) moieties. IncuCyte® image analysis software enables automated detection and selective quantitation of tumor cell death in real time.

General guidelines •• We recommend medium with low levels of riboflavin to reduce the green fluorescence background. EBM, F12-K, and Eagles MEM have low riboflavin (<0.2 mg/L). DMEM and RPMI have high riboflavin (>0.2 mg/L). •• Following cell seeding, place plates at ambient temperature (15 minutes for adherent cell lines and 45 minutes for non-adherent cell lines) to ensure homogenous cell settling. •• Remove bubbles from all wells by gently squeezing a wash bottle (containing 70-100% ethanol with the inner straw removed) to blow vapor over the surface of each well.

Required materials • IncuCyte® Caspase- 3/7 Apoptosis Reagent (Essen Bioscience Cat #4440) or • IncuCyte® Annexin V Green Reagent (Essen BioScience Cat #4642) or • IncuCyte® Annexin V Red Reagent (Essen BioScience Cat #4641) • Poly-L-ornithine (Sigma P4957), for non-adherent cells or • Fibronectin (Sigma F1141), for non-adherent cells • Flat bottom tissue culture plate (e.g., Corning 3595) • Target cells of interest (non-adherent target cells are required to be labeled with NucLight live-cell labeling reagent to enable tumor cell counting). • IncuCyte® NucLight Red Lentivirus Reagent (Essen BioScience Cat # 4476) • IncuCyte® NucLight Green Lentivirus Reagent (Essen BioScience Cat # 4475) • Immune (effector) cells of interest 96-well microplate (e.g., Corning® 3595)

•• After placing the plate in the IncuCyte® live-cell analysis system, allow the plate to warm to 37°C for 30 minutes prior to scanning.

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IncuCyte® Immune Cell Killing Assay

Immune cell killing of adherent tumor cells protocol 1

Seed target cells

Treat cells

3.3. Place the assay plate into the IncuCyte live-cell analysis system and schedule 24 hour repeat scanning: 3

a. Objective: 10x

Add immune cells

b. Channel selection: Phase Contrast + “Green” or “Red” depending on apoptosis reagent and target cell label used c. Scan type: Standard (2 images per well) d. Scan interval: Every 3 hours

Seed tumor cells (100 μL/ well, 1,000 to 3,000/well) into the 96-well plate. Optional: Target cells can be labeled with IncuCyte® NucLight Red live-cell labeling reagent (Essen BioScience 4476) to enable simultaneous tumor cell counting.

Aspirate the medium and add the Caspase 3/7 reagent or Annexin V reagent (50 μL/ well) and desired treatments (50 μL/well) at 4x final assay concentrations.

Add your choice of immune cells (100 μL/well, 10,000 to 30,000/well) to a 96-well plate.

Immune cell killing of non-adherent tumor cells protocol 1

Coat plate

Prepare treatments

Addition of target and effector cells

DAY 0: 1

Seed target cells

Coat plate surface to ensure even target cell coverage e.g. Poly-Lornithine solution.

1.1. Seed target cancer cells (100 μL per well) at an appropriate density into a 96-well flat-bottom plate such that by day 1 the cell confluency is approximately 20%. The seeding density will need to be optimized for each tumor cell line used; however, we have found that 1,000 to 3,000 cells per well are reasonable starting points.

Prepare Annexin V reagent (50 μL/well) and desired treatments (50 μL/well) at 4x final assay concentrations.

a. Target cell growth can be monitored using the IncuCyte® live-cell analysis system and confluence algorithm. b. Optional: Target cells can be labeled with NucLight Red live-cell labeling reagent (Catalog # 4475 or 4476) to enable simultaneous real-time counting of viable tumor cells.

DAY 1: 1

Prepare apoptosis reagent and treatments 2.1. Dilute apoptosis reagents, ensuring compatibility of target cell label and apoptotic marker, and treatments (e.g., T cell stimuli, antibodies, cytokines) at 4x final assay concentration in desired assay medium. a. If using caspase-3/7, dilute reagent to a concentration of 20 μM (1:250 dilution), sufficient for 50 μL per well. b. If using Annexin V reagents, solubilize Annexin V by adding 100 μL of complete medium or PBS. The reagents may then be diluted in complete medium containing at least 1 mM CaCl2 for a dilution of 1:50, sufficient for 50 μL per well. 2.2. Remove the cell plate from the incubator and aspirate off growth medium. 2.3. Add 50 μL each of the prepared apoptosis reagent and treatments from step 2.1 above. NOTE: For treatment controls, add 50 μL of assay medium.

Add treatments 3.1. Count chosen effector cells (e.g. T cells, PBMCs) and prepare a cell suspension at a density of 100,000 to 300,000 cells/mL (100 μL per well, 10,000 to 30,000 cells/ well). It is recommended that different target-to-effector cell ratios are tested (e.g., 1:5, 1:10). NOTE: Assay duration may be reduced by pre-activating the effector cells before addition to assay plate, however, this may require a higher initial seeding density of target cells. 3.2. Seed 100 μL of effector cells into the appropriate wells of the cell plate to achieve a final assay volume of 200 μL. Allow plates to settle on level surface at ambient temperature for 30 minutes.

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Coat plate 1.1. Coat a 96-well flat bottom plate with relevant coating matrix. We recommend coating with 50 μL of either 0.01% polyL-ornithine solution or 5 μg/mL fibronectin diluted in 0.1% BSA. Coat plates for 1 hour at ambient temperature, remove solution from wells, then allow plates to dry for 30-60 minutes prior to cell addition. Choice of coating will need to be determined prior to the assay for target cells of interest.

DAY 1: 2

Add NucLight labeled target cells (50 μL/well, 10,000 to 20,000/ well) and immune cells (50 μL/ well, 100,000 to 200,000/well) to a 96well plate.

Prepare apoptosis reagent and treatments 2.1. Prepare the following reagents in medium: a. Test materials (e.g. T cell stimuli, antibodies, cytokines; 50 μL per well, prepared at 4x final assay concentration). b. Apoptosis detection reagent (ensure compatibility of cell label and apoptotic marker), IncuCyte® Annexin V Reagent (Cat # 4641 or 4642): solubilize Annexin V by adding 100 μL of complete medium or PBS. The reagents may then be diluted in complete medium containing at least 1 mM CaCl2 for a dilution of 1:50 (4x final assay concentration, 50 μL per well. NOTE: Although either the IncuCyte® Annexin V or Caspase-3/7 reagents can be used to detect immune cell killing of target cells we recommend that the Annexin V reagent is used for non-adherent target cells. Non-adherent target and effector cell types can have very similar nuclear sizes negating the use of size filters to remove Caspase-3/7 labeled effector nuclei from the analysis. Additionally, we have observed raised levels of caspase 3/7 activity in some non-adherent cell types, particularly at higher confluency, which can interfere with the interpretation of immune cell driven target cell death. In our experience the Annexin V reagent labels fewer effector cells and provides lower non-specific background. 2.2. Add all prepared reagents to assay plate to achieve 100 μL per well.

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Add immune cells 3.1. Count labeled target cells and prepare a cell suspension at a density of 40,000 – 80,000 cells/mL (seed 50 μL per well, 10,000 to 20,000 cells/well). Target cells can be labeled with NucLight Red or Green live-cell labeling reagent (Cat # 4476 or 4475) to enable simultaneous real-time counting of viable tumor cells. 3.2. Count chosen effector cells (e.g. T cells, PBMCs) and prepare a cell suspension at a density of 400,000 – 800,000 cells/mL (50 μL per well, 100,000 to 200,000 cells/well). It is recommended that different target-to-effector cell ratios are tested (e.g. 1:5, 1:10). NOTE: Assay duration may be reduced by pre-activating the effector cells before addition to assay plate, however, this may require a higher initial seeding density of target cells. 3.3. Add target and effector cells to assay plate to achieve a final assay volume of 200 μL. Allow plates to settle on level surface at ambient temperature for 30 minutes. 3.4. Place the assay plate into the IncuCyte® instrument and schedule 24 hour repeat scanning: a. Objective: 4x b. Channel selection: Phase Contrast + “Green” and “Red” c. Scan type: Standard (2 images per well) d. Scan interval: Every 2–3 hours

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IncuCyte® Scratch Wound Assay For the measurement of invasion or migration into a wound region

This protocol provides an overview of our cell motility assay, which is suitable for the analysis of migration or invasion of adherent cell lines. This method utilizes our IncuCyte® WoundMaker tool to create 96 precise, uniform cell-free zones in cell monolayers cultured on IncuCyte® ImageLock 96-well plates. IncuCyte® Scratch Wound analysis software enables real time, automated measurement of label-free or dual fluorescence of cell migration and invasion in vitro.

General guidelines •• Following cell seeding, place plates at ambient temperature for 15 minutes to ensure homogenous cell settling. •• Do not leave any empty (dry) wells– these will damage the WoundMaker pins when creating the scratch. •• Remove bubbles from all wells by gently squeezing a wash bottle (containing 70-100% ethanol with the inner straw removed) to blow vapor over the surface of each well.

Required materials • Cell Migration/Invasion Kit (Essen BioScience Cat #4474), includes: – 96-well Cell Migration Software Application Module (Essen BioScience Cat #4400) – 96-pin IncuCyte WoundMaker Tool (Essen BioScience Cat #4563) – Two (2) Wound Maker Rinse Boats (Essen BioScience Cat # 5025-0191-A00) – Fifteen 96-well ImageLock plate (Essen BioScience Cat #4379) • IncuCyte 96-well Scratch Wound Cell Invasion Accessories (Essen BioScience Cat #4444), for invasion assay includes: – 2 x CoolBox 96F System plus 2 x CoolSink 96F – 1 x extra CoolSink 96F • Matrigel® (BD Cat#354234), for invasion assay

•• After placing the plate in the IncuCyte® live-cell analysis system, allow the plate to warm to 37°C for 30 minutes prior to scanning.

General guidelines For optimal assay quality, for both IncuCyte Scratch Wound cell migration and cell invasion assays it is recommended that cell density, the timing of the scratch wound (post cell plating) and the density of biomatrix material (if required) be investigated and optimized for different cell types. Cell Density: The most consistent wounds are generally made when the cell monolayer is at or very near to 100% confluence; typically seeding density will range from 10-50K cells per well. Timing of Scratch: Plating cells at the end of the day and wounding cells in the morning of the following day works well for many cell types plated on tissue culture plastic. If a

biomatrix material is being utilized, cells may adhere in just a few hours, and successful wounding may be possible on the same day of seeding. On occasion, cells will adhere too tightly, causing adhered cells debris after wounding, which blocks subsequent cell migration. Plating cells for shorter time periods, (e.g. 4 – 8h) can help improve the quality of the wounds. Biomatrix: Coating the well with a biomatrix material (e.g. Collagen-I) or poly-D-Lysine will typically enhance the timing and strength of cell attachment. It can allow cells to adhere more tightly to the substrate as opposed to each other, avoiding cell sloughing or the removal of sheets of cells.

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IncuCyte® Scratch Wound Assay

Migration Protocol 1

Coat Plate with ECM (optional)

Invasion Protocol 2

Coat plate surface to ensure cell attachment (e.g., Collagen-1).

Coat wells

Plate cells (100 μL/ well, 10,000-40,000 cells/well) and allow to adhere overnight.

Create wound area

Wound confluent cell monolayer using 96-well WoundMaker.

Add treatment

Coat plate with ECM (if required) 1.1. Coat a 96-well ImageLock plate with a thin layer (50 µL/well) of biomatrix. Gently rock the plate to ensure even coating of each well. 1.2. Depending on biomatrix used for coating, aspirate and wash coating from the wells prior to cell seeding.

Coat Plate with ECM (optional)

Coat plate surface with 100 mg/mL Matrigel (50 mL/well) to ensure cell attachment.

Add modulators of migration (100 μL/well).

DAY 0: 1

Seed cells

Plate cells (100 μL/well, 10,000-40,000 cells/well) and allow to adhere overnight.

Create wound area

Wound confluent cell monolayer using 96-well WoundMaker.

Add ECM + media containing treatment

Overlay cells with ECM (50 μL/well) ± treatment, polymerize, then overlay wells with media ± treatment (100 μL/well).

IMPORTANT:

In advance of invasion experiments it is important to have stored the Cool pack accessories at the correct temperatures for at least 4h: •• Coolbox x 2 (block with gelpack: -20°C), Coolsink 96F x2 (4°C), •• Coolsink 96F x 1 (37°C).

Seed cells

•• CoolBox M30 System (block with gelpack: -20 °C) with CoolRack (4°C).

2.1. Seed cells at a density of 10,000–40,000 cells/well (100 µL/well; 100,000–400,000 cells/mL stock) into each well of the coated 96-well ImageLock plate.

The Cool Packs are used to ensure close temperature control of Matrigel® in microplates. At 4-8°C, Matrigel is a viscous liquid. Polymerization will occur slowly at 4-8°C, and more rapidly when at room temperature or higher. For this reason, it is imperative to keep Matrigel solutions at 4-8°C during experimental set-up to avoid unwanted gelling. It is easier to handle low volume (<500μL) ECM solutions using pre-cooled (from a fridge), wide bore pipette tips or serological pipettes. We recommend sourcing a batch of Matrigel with a concentration of >9mg/mL and an endotoxin level of <1.5 (EU)/mL.

2.2. Allow the cells to settle at ambient temperature for 15 minutes, then place the plate into a 37°C incubator, 5% CO2 overnight or as pre-determined in assay optimization.

DAY 0: DAY 1: 3

Create wound

1.1. 1.1. Using a CoolSink M30 System with CoolRack, dilute Matrigel® stock to 100 μg/mL in culture media.

3.1. Carefully remove the ImageLock plate from the incubator, and use the WoundMaker (refer to Appendix I, Creating wounds section) to simultaneously create wounds in all wells.

Note: The day prior to coating the ImageLock plate, thaw a bottle of Matrigel®, packed in ice, overnight at + 4°C. When fully thawed, there should be no visible gel aggregates. If aggregates are present, replace the bottle on ice and thaw at + 4°C for a longer period of time. After thawing, chill ten 2 mL micro centrifuge tubes in the CoolSink M30 System (10min), and using a pre-cooled serological pipette, create 1 mL aliquots of Matrigel and store at -20°C.

3.2. After wounding, immediately aspirate the media from each well and carefully wash the cells twice with culture media (100 µL/well; with our without serum) or Dulbecco’s Phosphate Buffered Saline (dPBS), if desired. 4

Add treatments

1.3. Coat a 96-well well ImageLock plate with 50 µL/well of diluted Matrigel (100µg/mL). Gently rock the plate to ensure even coating of each well.

4.1. After washing, add 100 µl of culture media ± test material (e.g. small molecules, antibodies) to each well.

1.2. Place the plate in a 37°C incubator, 5% CO2 and allow the biomatrix material to polymerize for 2 hours.

4.2. Place the cell plate into the IncuCyte live-cell analysis system and allow the plate to warm to 37°C for 30 minutes prior to scanning.

Seed cells

a. Objective: 4x, 10x (recommended), or 20x

2.1. Remove plate from 37°C. Using a manual pipette, aspirate the Matrigel coating from the wells prior to cell seeding.

b. Channel selection: Phase Contrast (+ Fluorescence if analyzing cells with fluorescent labels)

2.2. Seed cells at a density of 10,000–40,000 cells/well (100 µL/well, 100,000–400,000 cells/mL stock) into each well of the coated 96-well ImageLock plate.

c. Scan type: Scratch Wound (Wide Mode optional for 10x, required for 20x) d. Scan interval: Every 1-3 hours 4.3. Wash and store the WoundMaker according to the wash protocol.

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Coat plate with ECM

2.3. Allow the cells to settle at ambient temperature for 15 minutes, then place the plate into a 37°C incubator, 5% CO2 overnight.

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The following steps should be performed in a biological safety cabinet: DAY 1: 3

Prepare biomatrix top layer, then create wound 3.1. On ice, prepare the biomatrix top layer by diluting Matrigel, typically 4-8 mg/mL, in cold culture media ± treatments. 3.2. Carefully remove the ImageLock plate from the incubator, and use the WoundMaker (refer to Appendix I, Creating wounds section) to simultaneously create wounds in all wells. 3.3. After wounding, immediately aspirate the media from each well and carefully wash the cells twice with culture media (100 µL/well). 3.4. After washing, add 100 µl of culture media, then cool the cell plate to 4°C for 5 minutes using the CoolSink and CoolBox 96F.

Add ECM and media ± treatments 4.1. Aspirate media from wounded ImageLock plate, and carefully overlay cells with 50µL of the Matrigel top layer (prepared in step 3.1). Remove any bubbles from the assay plate.

STEP 1

STEP 2

4.2. To polymerize the biomatrix top layer, warm the cell plate to 37°C by placing the plate onto a pre-warmed CoolSink inside the incubator.

•• Remove top of WoundMaker.

•• Insert plate (containing cells & media) into base plate holder.

•• Set top in empty wash boat.

•• Remove plate cover.

Creating wounds: Six simple steps

STEP 3

STEP 4

The WoundMaker is a 96-pin mechanical device designed to create homogeneous, 700-800 µm wide wounds in cell monolayers on IncuCyte® ImageLock 96-well microplates. The device is simple to use and wounds can be created in seconds.

•• Do not push down.

4.3. After 30 minutes, add additional media (100 µL/well ± treatments). Note: Leaving the plate to polymerize longer than 60 minutes will cause the biomatrix top layer to dehydrate. 4.4. Place the cell plate into the IncuCyte live-cell analysis system and allow the plate to warm to 37°C for 30 minutes prior to scanning. a. Objective: 4x, 10x (recommended), or 20x b. Channel selection: Phase Contrast (+ Fluorescence if analyzing cells with fluorescent labels) c. Scan type: Scratch Wound (Wide Mode optional for 10x, required for 20x) d. Scan interval: Every 2-3 hours 4.5. Wash and store the WoundMaker according to the wash protocol.

•• Replace pin block by guiding the rear dowels of pin block into the rear holes of the base plate.

•• Push and hold the black lever.

When used, stored and cleaned correctly, the WoundMaker should continue to provide clean, consistent wounds without damaging the cells or the underlying plastic or biomatrix. The WoundMaker is comprised of:

1 x 96 well pin block

1 x metal base

2 x 96 wash boats

STEP 5

STEP 6

•• Lift pin block while continuing to hold the black lever down.

•• Replace plate cover. •• Wash wells (up to two washes). •• Add treatment conditions. •• Put into the IncuCyte and start acquiring data and images.

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IncuCyte® Chemotaxis Cell Migration Assay

Optimization considerations

For the detection of chemotactic-induced cell migration

The IncuCyte® Chemotaxis Cell Migration Assay approach allows for automated imaging and analysis of cell migration using an optically clear membrane that allows for 96-well kinetic throughput. Using phase-contrast and/or fluorescent imaging in combination with IncuCyte® integrated metrics, we are able to precisely quantify the chemotactic response of adherent or nonadherent cell types. The assay method does not require labeling for quantification, is sensitive to surface integrin signaling, sustains a linear gradient over several days and allows for direct visualization of cell migration.

Required materials • IncuCyte® ClearView 96-well Cell Migration Plate (EssenBioscience cat # 4582) • IncuCyte® ClearView Reservoir Plate (EssenBioScience cat# 4600)

commonly used to promote cell migration. Refer to “IncuCyte® ClearView Plate Coating Protocol” for recommended coating procedures. Chemoattractant: The chemoattractant concentration required for an optimal assay should be determined through experimentation. Based on published literature and/or experience, we recommend testing the expected EC50 concentration as well as testing concentrations 1-2 logs above and below the EC50. Medium: We have found that including a reduced amount of serum (0.1%–2.5%) in the assay buffer allows the cells to attach to the surface and move, while not affecting their directional migration. If cells look unhealthy, an experiment should be designed to increase serum or growth factor levels until cells are healthy enough to attach and move. Adding Insulin-TransferrinSelenium (ITS) to the assay medium is another way to make cells healthier if minimizing serum is required.

• IncuCyte® Chemotaxis Software Module (EssenBioScience cat# 9600-0015)

Protocol

General guidelines It is important to read the protocol in its entirety prior to initiating a chemotaxis assay. The IncuCyte® live-cell analysis system relies on images to process data; thus it is important to avoid bubbles and follow our protocol recommendations to achieve superior assay performance and imaging. We recommend the following techniques to eliminate bubbles from your experiment: •• Reverse-pipette at the coating step and when adding cells to the insert. Reverse pipetting reduces the risk of splashing or bubble formation. In reverse pipetting, the volume aspirated into the tip is larger than the volume delivered to the receiving vessel. −− Press the plunger to the second stop. −− Dip the pipette-tip into the solution. −− Release the plunger until the starting position has been reached. −− Move the pipette-tip to the receiving vessel. −− Dispense the liquid by pressing the plunger to the first stop. SOME LIQUID WILL REMAIN IN THE TIP. −− Repeat steps 2–5 until throughout the plate.

Cells in the IncuCyte® Chemotaxis Cell Migration Assay are required to move toward a chemoattractant gradient across the membrane surface and through a pore. As a result, cells must be maintained in a healthy state on a biologically relevant surface, in order to facilitate cell movement. Cell surface coatings, chemoattractant and assay medium formulations are parameters that should be optimized in order to achieve superior assay performance. Prior to starting optimization assays, we recommend a thorough review of literature in order to become familiar with standard culturing techniques as well as Boyden chamber assay conditions for the cell type of interest. Although not all techniques will translate into the IncuCyte® Chemotaxis Cell Migration Assay, they will give guidance for an optimization strategy. Coating: Migrating cells require interactions with the substrate in order to move. Collagen I, collagen IV, gelatin (Attachment Factor), fibronectin, Matrigel®, and protein G/ICAM surface coatings are

•• Triturate with an additional cell volume or reduced volume setting (e.g., 60 μL cell volume added, mix by reverse-pipetting up and down with 30 μL) to dislodge bubbles that may have been trapped at the membrane-insert interface. Perform this immediately after cell addition. •• Remove bubbles at the liquid surface by gently squeezing a wash bottle containing 100% ethanol, with the inner straw removed, to blow vapor over the surface of each well. •• Gently place the insert into the pre-filled reservoir at a slight angle to allow air to move upwards across the membrane.

Coat insert (optional)

Coat membrane with extracellular matrix (ECM): 20 μL (insert side) and 150 μL (reservoir side).

Harvest and seed cells

Seed cells (*60 μL/ well, 1,000 or 5,000/ well) into the IncuCyte® ClearView insert. *If assay requires addition of treatment, seeding volume should be reduced to 40 μL.

Treat cells (optional)

Prepare 3x concentrations of treatment and add 20 μL to appropriate wells. Mix by triturating a 30 μL volume.

Allow cells to settle

Place on level surface and allow cells to settle at ambient temperature for 15 to 60 minutes.

Add chemoattractant

Add 200 μL of chemoattractant or controls to reservoir plate. Place the insert containing cells into the prefilled plate, and image in IncuCyte® system.

DAY 0: 1

Coat insert (optional) Some cell lines may require the addition of an extracellular matrix protein (e.g., 5 μg/mL fibronectin) to promote light cell adherence and provide the necessary integrins for cell motility. Refer to Table 1 for cell-line specific coating recommendations. 1.1. Under sterile conditions prepare coating matrix at desired concentration. 1.2. Using reverse pipetting, aliquot 150 μL of the prepared matrix into the reservoir wells and 20 μL into the insert wells. 1.3. Incubate according to manufacturer’s recommendations 1.4. If required, aspirate and wash coating from the reservoir and insert prior to cell seeding

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IncuCyte® Chemotaxis Cell Migration Assay

Harvest and seed cells

Validated cell lines

2.1. Harvest cells using suitable dissociation solution.

NOTE: Detailed protocols are available for some of the validated cell lines by clicking on the cell type name.

2.2. Spin down cells and resuspend in appropriate assay medium (i.e. growth medium with reduced FBS, typically 0.5% FBS). 2.3. Determine cell concentration and prepare cell seeding stock to achieve 1,000 or 5,000 cells per well at a 60 μL volume, or, if using modulators of chemotaxis (refer to step 3 below), the seeding volume will be reduced to 40 μL.

THERAPEUTIC AREA

NOTE: The seeding density will need to be optimized for each cell type used; however, we have found that 1,000 cells per well for adherent cell types and 5,000 cells per well for non-adherent cell types are reasonable starting points.

CELL TYPE

ASSAY MEDIUM

5,000

ng/mL

50 μg/mL Matrigel + 10% FBS in assay medium (for clustered cell migration)

45-60

SDF-1α (12. 5 – 125)

SDF-1α (100– 1,000)

45-60

SDF-1α (3.1 – 200) CXCL11 (12.5 – 800) RANTES (16)

SDF-1α (24.8 – 1,600) CXCL11 (103.8 – 6640) RANTES (125) C5a (21.6 – 15,770) fMLP (0.6 – 437.6) IL-8 (13.4 – 10,000) LTB4 (0.5 – 336.5)

RPMI + 0.5% FBS

T Cells

RPMI + 0.5% FBS

20 μg/mL Protein G followed by 5 μg/mL ICAM

Neutrophils

RPMI + 0.5% HSA

50 μg/mL Matrigel + 10% FBS in assay medium

5,000

45-60

C5a (2.6 – 1,900) fMLP (1.4 – 1,000) IL-8 (1.5 – 1120) LTB4 (1.4 – 1,000)

CCRF-CEM

RPMI + 50% Human Serum

20 μg/mL Protein G followed by 5 μg/ mL ICAM OR 5 μg/mL Fibronectin

5,000

45-60

CCL22 (1 – 100) SDF-1α (1 – 100)

CCL22 (8.1 – 810) SDF-1α (8 – 800 nM)

THP-1

RPMI + 0.5% dialyzed FBS

5,000

45-60

3.1. Prepare 3x concentrations of treatment and add 20 μL to the insert wells containing cells immediately after cell seeding.

50 μg/mL Matrigel + 10% (dialyzed FBS in assay medium)

RANTES (08 – 780) MIP-1α (0.8 – 780) MCP-1 (12 – 8,600)

RANTES (0.1 – 100) MIP-1α (0.1 – 100) MCP-1 (1.4 – 1,000)

Differentiated THP-1

RPMI + 0.5% FBS

5 μg/mL Fibronectin in D-PBS

2,500

**45-60

C5a (0.5 – 2,000)

C5a (4.2 – 16,600)

3.2. Using a 30 μL volume, triturate the cells to appropriately mix the treatment, so cell exposure during pre- treatment is at 1x.

Primary Macrophages

RPMI + 0.5% FBS

50 μg/mL Matrigel + 10% FBS in assay medium

2,000

15-30

C5a (10 – 1000)

C5a (83 – 8,300)

HUVEC

EBM-2 + ITS + 0.25% FBS With #supplements

5 μg/mL Fibronectin + 0.1% BSA in D-PB

1,000

VEGF (0.01 – 2.6)bFGF (0.02 – 17.4)FBS (0.4 – 10%)

VEGF (0.4 – 100) bFGF (0.4 – 300) FBS (0.4 – 10%)

HMVEC

EBM-2 + 0.5% FBS + ITS with #supplements

10 μg/mL Fibronectin + 0.1% BSA in D-PB

1,000

bFGF (0.06 – 5.8) FBS (10%)

bFGF (0.01-0.1) FBS (10%)

HT 1080

F12 + ITS*

None

1,000

MDA-MB-231

DMEM + 2.5 FBS

None

1,000

EGF (0.2 – 1.6) FBS (10%) – slight response

EGF (1 – 10n) FBS (10%) – slight response

MCF10a

DMEM/F12 + 10 μg/mL insulin hydrocortozone + 100ng/mL cholera toxin

5 μg/mL Collagen IV in 0.05M HCl

1,000

EGF (0.2 – 17,200)

EGF (0.1 – 1,000)

RECOMMENED SEEDING STOCK VOLUME (mL)

1,000

25,000

5,000

125,000

1,000

16,666.70

5,000

83,333.30

IMMUNOLOGY

CELL SEEDING STOCK (CELLS/mL)

Non-adherent

FINAL CELL DENSITY/WELL

Treat cells (optional)

Harvest and seed cells

NOTE: If treatments were added to an adherent cell type, we recommend a continued pre-incubation with inhibitors at 37°C for 30 minutes. Chemoattractant addition and imaging 5.1. Add 200 μL of desired chemoattractant or control to the appropriate wells of the reservoir plate. 5.2. Carefully transfer the insert into the pre-loaded reservoir plate. Be careful not to introduce bubbles which can become trapped below the membrane when placing the insert into the pre-filled reservoir plate. 5.3. Place the ClearView Cell Migration plate into the IncuCyte® system and allow the plate to warm to 37°C for at least 15 minutes. After 15 minutes, carefully wipe away any condensation that may have accumulated on top of the plate lid or on the bottom of the reservoir plate. Schedule 24 hour repeat scanning:

Adherent

4.1. Place the plate onto a level surface and allow the cells to settle at ambient temperature for 15 minutes (adherent cell types) to 45-60 minutes (non-adherent cell types).

ONCOLOGY

VASCULAR

Cell treatment should be prepared prior to cell seeding. It is critical to add modulators immediately following cell seeding in order to ensure appropriate cell exposure and homogenous cell settling.

5,000

CHEMOATTRACTANT (CONCENTRATION RANGE TESTED)

Jurkat

SEEDING VOLUME

SEED DENSITY (CELLS/WELL)

CELL SETTING TIME (MIN)

5 μg/mL Fibronectin + 0.1% BSA in D-PBS

2.4. Using reverse pipetting, add cells (if not using inhibitors of chemotaxis, go directly to step 4).

COATING (20 ΜL TOP/ 150 ΜL BOTTOM

FBS (0.01 – 10)%

* Insulin-Transferrin-Selenium (Life Technologies Cat# 41400-045) # Supplements: EGM-2 singlequot bullet kit (LONZA CC-4176) with growth factors removed and reduced FBS ** Cell settling time pre-differentiation

a. Objective: 10x b. Channel selection: Phase Contrast (+“Red” if fluorescent labeled cell line is used) c. Scan type: Chemotaxis d. Scan interval: Every 1 to 2 hours

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Protocol 1

Prime insert

Harvest and seed cells

Polymerize matrix

Media addition

Add chemoattractant

For the detection of chemotactic-induced cell invasion

The IncuCyte® Chemotaxis Cell Invasion Assay approach allows for automated imaging and analysis of cell invasion using an optically clear membrane that allows for 96-well kinetic throughput. Measurements of chemotactic cell invasion can be made using nuclear labeled or unlabeled cells, however, we recommend labeling cells with a live-cell nuclear label (e.g. IncuCyte® NucLight reagents) to ensure optimal performance. Using IncuCyte® integrated metrics, we are able to precisely quantify the chemotactic response of adherent types, with direct visualization of cell invasion.

Prime membrane by adding 150 L of D-PBS to the reservoir of a pre-chilled ClearView Cell Migration plate. Incubate plate for 20 minutes at 4°C.

Required materials • IncuCyte® ClearView 96-well Chemotaxis Plate (Essen Bioscience cat # 4582) • IncuCyte® ClearView Reservoir Plate (Essen BioScience cat# 4600)

Place ClearView plate on a pre-warmed CoolSink at 37°C. Allow matrix with embedded cells to polymerize for 30-60 minutes.

Add 40 μL assay medium on top of the polymerized matrix:cell layer.

Add 200 μL of desired chemoattractant or controls to appropriate wells of the reservoir plate. Place the insert into the pre-filled and image in IncuCyte® system.

• IncuCyte® Cell Invasion Kit (Essen BioScience cat# 4444) • Cultrex® 3-D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract (Trevigen 3445-005-01) optional

DAY 0: 1

1.2. Add 150 μL of D-PBS (4°C) to each reservoir well of the pre-chilled ClearView chemotaxis plate. Replace the ClearView insert and allow the membrane to

• IncuCyte® Chemotaxis Software Module (Essen BioScience cat# 9600-0015)

General guidelines

Avoiding bubbles

Reagent temperature control

The IncuCyte system relies on images to process data; thus, it is important to avoid bubbles and follow our protocol recommendations to achieve superior assay performance and imaging. We recommend the following techniques to eliminate bubbles from your experiment: •• Reverse-pipette at the coating step and when adding cells to the insert. Reverse pipetting reduces the risk of splashing or bubble formation. In reverse pipetting, the volume aspirated into the tip is larger than the volume delivered to the receiving vessel.

•• It is important to keep close temperature control of biomatrix materials such as Collagen-1 and basement membrane extract (BME) to prevent unwanted gelling.

Prime insert 1.1. Pre-cool a ClearVew Chemotaxis plate in a CoolBox system containing a frozen cold pack and CoolSink plate (4°C) for 5 minutes

• Cultrex® Rat Collagen I (Trevigen 3440-100-01) optional

−− Press the plunger to the second stop. −− Dip the pipette-tip into the solution. −− Release the plunger until the starting has been reached. −− Move the pipette-tip to the receiving vessel. −− Dispense the liquid by pressing the plunger to the first stop. SOME LIQUID WILL REMAIN IN THE TIP. −− Repeat steps 2–5 until throughout the plate. •• Triturate with an additional cell volume or reduced volume setting (e.g., 60 µL cell volume added, mix by reversepipetting up and down with 30 µL) to dislodge bubbles that may have been trapped at the membrane-insert interface. Perform this immediately after cell addition.

Prepare cells at 50,000 cells/mL in extracellular matrix. Dispense 20 μL/well of cell:matrix suspension (1,000 cells/well) into insert. Centrifuge for 3 minutes at 50x g.

Harvest and seed cells 2.1. While the ClearView plate is priming, prepare your biomatrix reagent at the desired working concentration as per the manufacturer’s instructions. Prepare a large dead volume to ensure available biomatrix for transfer to the assay plate (e.g., prepare 4 mL biomatrix solution to provide 20 μL per insert well). NOTE: The required biomatrix density will be dependent on the matrix and cell types used. For HT-1080 cells we recommend BME (5 mg/mL) diluted in assay medium or Collagen I (1 mg/mL) diluted in neutralizing buffer (DMEM, Sigma D2429, + 7.5 g/L sodium bicarbonate + 0.004 g/L folic acid + 1% GlutaMax). When preparing collagen I it is important that it is properly neutralized to ensure cell health is maintained and gelling is uniform.

•• The IncuCyte® Cell Invasion Kit (Cat. No. 4444) includes a specialized CoolBox™ system to ensure the temperature of your assay plate and biomatrix materials are maintained between 4-8°C – preventing premature polymerization and eliminating edge effects. Crushed ice can be used as an alternative however non-uniform cooling can lead to assay variability.

2.2. Harvest your cells and resuspend the pellet in the biomatrix solution. Cell density will need to be optimized for each cell type used; however, we have found that 50,000 cells/mL (1,000 cells per well) is a reasonable starting point. Calculation: 50,000 cells/mL x 0.02 mL/well = 1,000 cells/well NOTE: Some cell types may require reduced exposure to Fetal Bovine Serum (FBS) before initiating the transmembrane invasion assay (e.g., HT-1080s starved in F12 + Insulin-Transferrin-Selenium for ~20 hours).

•• When handling cells that have been embedded in biomatrix material, ensure that all steps of cell handling is performed between 4-8°C, utilizing consumables that have been prechilled (e.g., a pre-cooled reservoir boat during cell seeding).

2.3. Seed cells (20 μL per well, 1000 cells per well) into every well of the cooled ClearView insert plate. 2.4. Centrifuge the ClearView plate in a cooled centriguge for three minutes at 50 x g. 3

Polymerize matrix 3.1. Place the ClearVIew plate at 37°C on a pre-warmed CoolSink and allow the biomatrix to polymerize for 30 -60 minutes.

•• Remove bubbles at the liquid surface by gently squeezing a wash bottle containing 100% ethanol, with the inner straw removed, to blow vapor over the surface of each well.

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Media addition 4.1. Gently add 40 µL of assay medium ± modulators of invasion on top of the biomatrix:cell layer in the insert wells. NOTE: If adding modulators of invasion, the working concentration should be 1.5x the desired final concentration to account for the volume of the biomatrix: cell layer

Chemoattractant addition and imaging 5.1. Add 200 µL of desired chemoattractant or control to the appropriate wells of a second reservoir plate. 5.2. Carefully transfer the insert into the pre-loaded reservoir plate. Be careful not to introduce bubbles which can become trapped below the membrane when placing the insert into the pre-filled reservoir plate. 5.3. Place the ClearView Cell Migration plate into the IncuCyte® system and allow the plate to warm to 37°C for at least 15 minutes. After 15 minutes, carefully wipe away any condensation that may have accumulated on top of the plate lid or on the bottom of the reservoir plate. Schedule 24 hour repeat scanning: a. Objective: 10x b. Channel selection: Phase Contrast +“Red” or “Green” channels selected (dependent on the fluorescent label used) c. Scan type: Chemotaxis d. Scan interval: Every 1 to 2 hours

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Chemotatic Transendothelial Migration Assay For quantification of leukocyte migration across an endothelial monolayers

This protocol is intended for the measurement of transendothelial migration by leukocytes. This method utilizes the IncuCyte® ClearView 96-well Chemotaxis plate and the IncuCyte live-cell analysis system for image-based measurements of diapedesis.

Required materials • Leukocytes (e.g. activated primary T-cells or neutrophils) • Endothelial cells (e.g. HUVEC or EA.hy9226) • Endothelial growth medium

General guidelines

• Assay medium • Collagen Type I Rat Tail (BD Biosciences 354236) or • Fibronectin (Sigma Aldrich F1141)

• Acetic Acid • D-PBS -/- (w/o Ca2+, Mg2+, Life Technologies 10010) – used in Fibronectin coating step • D-PBS +/+ (with Ca2+, Mg2+, Life Technologies 14040) – used in monolayer wash step • IncuCyte® ClearView 96-Well Chemotaxis Plate (Essen 4582 or 4599)

−− Press the plunger to the second stop. −− Dip the pipette-tip into the solution. −− Release the plunger until the starting has been reached. −− Move the pipette-tip to the receiving vessel. −− Dispense the liquid by pressing the plunger to the first stop. SOME LIQUID WILL REMAIN IN THE TIP. −− Repeat steps 2–5 until throughout the plate. •• Triturate with an additional cell volume or reduced volume setting (e.g., 60 µL cell volume added, mix by reverse-pipetting up and down with 30 µL) to dislodge bubbles that may have been trapped at the membrane-insert interface. Perform this immediately after cell addition. •• Remove bubbles at the liquid surface by gently squeezing a wash bottle containing 100% ethanol, with the inner straw removed, to blow vapor over the surface of each well.

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Chemotatic Transendothelial Migration Assay

Protocol 1

Coat insert

4 2

Seed endothelial cells

Seed leukocytes

Add chemoattractant

Add chemoattractant 4.1. Using a manual multi-channel pipette, add 200 µL of the chemoattractant and control medium to the appropriate wells of the second reservoir plate. 4.1. Carefully transfer the insert plate containing the cells into the pre-filled second reservoir plate containing assay medium ± chemoattractant. 4.1. Place the IncuCyte® ClearView cell migration plate into the IncuCyte® live-cell analysis system and allow the plate to warm to 37°C for at least 15 minutes. After 15 minutes, wipe away any condensation that remains on the outside of the plate lid or bottom of the reservoir. 4.1. In the IncuCyte® software, schedule 24 hour repeat scanning (10x) for every 30 minutes.

Coat membrane with extracellular matrix (ECM): 20 μL (insert side) and 150 μL (reservoir side).

Seen endothelial cells (60 μL/well, 6,000 cells/ mL) into the IncuCyte ClearView insert. Allow monolayer to form overnight.

Seed leukocytes (60 μL/ well, 5,000 cells/mL) onto endothelial monolayer.

Add 200 μL of chemoattractant or controls to reservoir plate. Place the insert into the prefilled reservoir plate and image in IncuCyte© system.

a. Scan Type: Chemotaxis b. Objective: 10x c. Channel selection: Phase Contrast +“Red” or “Green” channel if leukocytes are fluorescently labeled Leukocyte and endothelial cells that have been validated with the TEM and recommended growth and assay media

DAY 0: 1

Coat insert 1.1. Prepare extracellular matrix coating of either 50 µg/mL collagen diluted with 0.02N acetic acid or 5 µg/mL fibronectin diluted with D-PBS (-/-).

LEUKOCYTE

ENDOTHELIAL CELLS

ENDOTHELIAL GROWTH MEDIUM

Jurkat with

HUVEC

EGM-2

EBM-2 + 2% FBS with 5 µg/mL fibronectin #supplements or 50 µg/mL collagen

HUVEC

EGM-2

EBM-2 + 2% FBS with 5 µg/mL fibronectin #supplements or 50 µg/mL collagen

EA.hy926

DMEM + 10% dialyzed FBS

RPMI + 0.5% dialyzed FBS

1.2. Aliquot 150 µL of coating solution into the reservoir. Gently place the insert into the reservoir and pipette 20 µL of the fibronectin, or collagen, solution into the insert. 1.3. Incubate for 1 hour at ambient temperature 2

Create endothelial cell monolayer 2.1. During incubation, harvest and count endothelial cells and prepare a cell seeding stock of 100,000 cells/mL in full growth medium.

Primary T-cells

ASSAY MEDIUM

COATING (20 µL TOP/150 µL BOTTOM)

SEEDING DENSITY ENDOTHELIAL: LEUKOCYTE (CELLS/WELL)

6,000 : 5,000

50 µg/mL collagen

EBM-2 + 2% FBS with 5 µg/mL fibronectin #supplements or 50 µg/mL collagen #supplement includes gentamycin, hydrocortisone, and ascorbic acid Neurophils with

HUVEC

EGM-2

2.2. Aspirate the coating from the reservoir plate and replace with 200 µL of D-PBS (-/-) and gently return the insert into to the reservoir plate. 2.3. To the insert, directly add 60 µL D-PBS (-/-) to the inserts containing coating, then aspirate the entire volume. 2.4. Immediately seed 60 µL of the endothelial seeding stock in growth medium using a multi-channel pipette into every well of the insert plate (60 µL per well, 6,000 cells per well). Calculation: 100,000 cells/mL x 0.06 mL = 6,000 cells per insert well. 2.5. Allow the cells to settle at ambient temperature on a level surface for 15 minutes. 2.6. Place the Chemotaxis plate containing cells at 37°C and incubate for 24 hours DAY 1: 3

Seed leukocytes 3.1. After the endothelial monolayer has formed, gently wash the monolayer 2x with D-PBS (+/+), using partial washes NOTE: It is important not to disrupt the monolayer. It is recommended to gently remove about half of the growth medium then add 60 µL D-PBS for both washes. At the final wash step, remove as much of the medium/D-PBS as possible without disrupting the monolayer. 3.2. Prepare leukocyte cell seeding stock at 83,333 cells/mL in appropriate assays medium. 3.3. Using a manual multi-channel pipette and reverse pipetting technique, seed 60 µL of the leukocytes seeding stock (5,000 cells per well) into every well of the insert plate, being careful not to disrupt the endothelial monolayer. Calculation: 83,333 cells/mL x 0.06 mL = 5,000 cells per insert well. 3.4. Centrifuge the chemotaxis plate for 3 minutes at 50 x g in order to quickly bring the leukocytes to the monolayer surface. Alternatively, if centrifugation is not possible, allow the leukocytes to settle on the endothelial monolayer at ambient temperature for 45-60 minutes.

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IncuCyte® Antibody Internalization Assay

Preparation of IncuCyte® Antibody Internalization Assay 1

Seed Target target cells of interest 1.1 Harvest cells of interest and determine cell concentration (e.g. trypan blue + hemocytometer).

a. Add test antibody at 2X the final antibody concentration. We suggest optimizing the assay by testing a final concentration of 4 ug/ml of test antibody (e.g., 2X working concentration=8 ug/ml).

1.2 Prepare cell seeding stock in target cell growth media to achieve 40-50% confluence after 2-6 h. Suggested starting range 5,000-30,000 cells/well (depends on cell type used). NOTE: The seeding density will need to be optimized for each cell type. For non-adherent cell types a well coating may be required e.g. Poly-L-ornithine (PLO, Sigma P4957) to maintain even cell coverage in well (see IncuCyte Cell Proliferation Assay protocol on www.Essenbioscience.com for details).

This protocol describes a solution for the measurement of antibody internalization by cells expressing antigen of interest. This method utilizes the IncuCyte® FabFluor-pH Red Antibody Labeling reagent and the IncuCyte® live-cell analysis system for image-based fluorescent measurements of antibody internalization. Required materials

Recommended materials

• IncuCyte® FabFluor-pH Red Antibody labeling reagent (Essen BioScience cat #4722 human, cat #4723 mouse IgG1, cat #4737 rat , cat #4750 mouse IgG2a, or cat #4751 mouse IgG2b).

It is strongly recommended that a positive and negative control is run alongside test antibodies and cell lines. CD71, which is an anti-human mouse antibody, is recommended as a positive control for the mouse Fab.

• Test antibody of interest containing human, mouse, or rat Fc region (at known concentration).

• Anti-CD71, clone MEM-189, IgG1 e.g. Sigma

• Target cells of interest.

SAB4700520-100UG

• Target cell growth media.

• Anti-CD71, clone CYG4, IgG2a e.g. Biologend 334102

• PBS (w/o Ca2 /Mg2 , Life Tech 14190).

• Isotype controls, depending on isotype being studied

• 96-well flat bottom microplate (e.g. Corning® 3595) for imaging. • 96-well round black round bottom ULA plate (e.g. Corning® 45913799) or amber microtube (e.g. Cole parmer® MCT150-X) for conjugation step.

1.4 Remove bubbles from all wells by gently squeezing a wash bottle (containing 70-100% ethanol with the inner straw removed) to blow vapor over the surface of each well.

Seed cells

Label test antibody

NOTE: Depending on cell type, plates can be used in assay once cells have adhered to plastic and achieved normal cell morphology e.g. 2-3 hr for HT1080 or 1-2 h for nonadherent cell types. Some cell types may require overnight incubation.

– Mouse IgG2a e.g. Biolegend 401501 – Mouse IgG2b e.g. Biolegend 400322

Add to cells

Live-cell Fluorescent imaging

Labeling of test antibody 2.1 Rehydrate IncuCyte® FabFluor-pH Red Antibody Labeling reagent with 100 µL sterile water to result in a final concentration of 0.5 mg/mL. NOTE: A 1:3 molar ratio of test antibody to IncuCyte® FabFluor-pH Red reagent is recommended. The labeling reagent is a third of the size of a standard antibody, so equal mg/ml quantities will produce a 1:3 molar ratio of test antibody to labeling Fab.

Cell Seeding Seed cells (50 µL/well, 5,00030,000 cells/well), into 96-well plate and leave to adhere (224 h, depending on cell type).

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IncuCyte® FabFluorlabeled Antibody Addition

Automated Imaging andQuantitative Analysis

Add antibody-FabFluor mix (50 µL/well) to cell plate.

Capture images every 15-30 minutes (10x or 20x) in IncuCyte® for 24-48 hours. Analyze using integrated software.

NOTES: If performing a range of concentrations of test antibody e.g. concentration response-curve, it is recommended to create dilution series post conjugation step in media to ensure consistent molar ratio labeling. We strongly recommend the use of both a negative and positive control antibody (see Recommended Materials above). 3

1.5 Allow cells to settle on a level surface for 30 minutes at room temperature, then place in IncuCyte® live-cell analysis system to monitor cell confluence in well.

– Mouse IgG1, e.g. R&D Systems, MAB002 or Biolegend 400124

Quick Guide

c. Add media to bring the total volume to 50 ul/well. Triturate to mix.

1.3 Using a multi-channel pipette, seed cells (50 µL per well) into a 96-well flat bottom microplate. Lightly tap plate side to ensure even liquid distribution in well.

b. Add IncuCyte®FabFluor-pH Red Antibody Labeling reagent at 2X the final concentration. We suggest optimizing the assay by testing a final concentration of 4 ug/ml of IncuCyte FabFluor-pH Red Antibody (e.g., 2X working concentration=8 ug/ml).

This reagent is light sensitive. It is advised to keep in amber tubes or foil wrapped tubes. Remaining re-hydrated reagent can be aliquoted and stored at -80°C (avoid freezing and thawing, stable for > year). 2.2 Mix test antibody with dilute IncuCyte®FabFluor-pH Red Antibody Labeling reagent and target cell growth media in a black round bottom microplate or amber tube to protect from light (50 ul/well).

Add IncuCyte FabFluor-pH Red reagent to cells 3.1 Remove cell plate from incubator. 3.2 Using a multi-channel pipette, add 50 µL of labeled antibody to required test wells, remove any bubbles and immediately place plate in IncuCyte® live-cell analysis system.

Acquire images and analyze 4.1 In the IncuCyte® software, schedule 24 hour repeat scanning for every 15-30 minute (depending on speed of internalization signal). a. Scan on schedule, standard. b. Channel selection: select “phase” and “red” c. Objective: 10x or 20x depending on cell types used, generally 10x is recommended for adherent cells, and 20x for non-adherent or smaller cells. NOTE: If trying to achieve rapid first image acquisition, scheduling can be set up on the instrument and no start time of scan attached prior to addition of reagents to plate. The scan time can then set once the plate is placed in the instrument. By maintaining all reagents at 37°C prior to plate addition there is reduced risk of condensation formation on the lid and therefore no need for plate warming before first image acquisition.

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4.2 To generate the metrics, user must create an Analysis Definition suited to the cell type, assay conditions and magnification selected. 4.3 Select images from a well containing a positive internalization signal and an isotype control well (negative signal) at a time point where internalization is visible. 4.4 In the Analysis Definition: a. Set up the mask for the phase confluence measure with red channel turned off. b. Red channel turned on: Exclude background fluorescence from the mask by using the background subtraction feature. The feature “Top-Hat” will subtract local background from brightly fluorescent objects within a given radius; this is a useful tool for analyzing objects which change in fluorescence intensity over time.

i. The radius chosen should reflect the size of the fluorescent object but contain enough background to reliably estimate background fluorescence in the image; 20-30 μm is often a useful starting point. ii. The threshold chosen will ensure that objects below a fluorescence threshold will not be masked. iii. Choose a threshold in which red objects are masked in the positive response image but low numbers in the isotype control, negative response well. For a very sensitive measurement, for example, if interested in early responses, we suggest a threshold of 0.2. NOTE: The adaptive feature can be used for analysis but may not be as sensitive and may miss early responses. If interested in rate of response, Top Hat maybe preferable.

Example calculation for antibody labeling using positive control anti-CD71 at 1 mg/mL stock concentration 1. Determine final assay concentration of test antibody — 4 µg/mL for anti-CD71 is recommended for positive control wells. Working concentration will be 2X or 8 µg/mL. 2. Determine volume of labeled antibody required at 2X final assay concentration (dilution of 1:2 recommended upon addition to cells): [# wells] x 50µL (plus additional required to prepare dilution series if desired). For 8 replicates of highest concentration plus 8 replicates of 1:2 dilution of labeled test antibody: 8 x 50 µL x 1.5 = 600 µL minimum (650µL used for this example) 3. Calculate volumes of test antibody, IncuCyte FabFluor-pH Red reagent, and media required to provide 2X final assay concentration of labeled test antibody.

• volume of IncuCyte FabFluor: [volume of test antibody] µL X [stock concentration of test antibody] mg/mL / [stock concentration of test FabFluor] mg/mL 5.2 µL x 1 mg/mL / 0.5 mg/mL = 10.4 µL NOTE: IncuCyte FabFluor-pH Red reagent is a third of the molecular weight of a standard antibody, so equal volumes of equal mg/ mL quantities will produce a 1:3 molar ratio of test antibody to FabFluor as MW of a typical antibody is ~3x of FabFluor. In this case, the stock concentration in mg/mL of the test antibody is twice that of FabFluor, thus the FabFluor volume should be 2X the volume of the test antibody. • Determine volume of media: [total volume] – [test antibody volume] – [FabFluor volume] = 634.4 µL

• Determine volume of test antibody: [total volume] µL x [working concentration test antibody] µg/mL / [stock concentration test antibody] mg/mL /1000 650 µL x 8 µg/mL / 1 mg/mL / 1000 = 5.2 µL

Analysis Guidelines As the labeled antibody is internalized into the acidic environment of the lysosome the area of fluorescence and intensity inside the cells increases. This can be reported in two ways: i. An increase in fluorescence area (“total object area” or “red object confluence”). Suggested metric: Analyze using the “Total Red Object Area (μm2/well)”. 124

ii. An increase in intensity, integrated over the area of detectable fluorescence (“Total Integrated Intensity”). Suggested metric: Analyze using “Total Red Object Integrated Intensity (RCU x μm2/well)” metrics.

IncuCyte® Live-Cell Immunocytochemistry Assay For cell surface marker analysis

This protocol describes a solution for measuring immunocytochemistry in live-cells expressing a surface antigen of interest. The method utilizes the IncuCyte® FabFluor-488 Antibody Labeling Reagent in combination with IncuCyte OptiGreen and the IncuCyte® live-cell analysis system for image-based fluorescent measurement enabling live-cell immunocytochemistry. Required materials

Recommended materials

• IncuCyte® FabFluor-488 Antibody Labeling Reagent plus IncuCyte® Opti-Green suppressor (Sartorius cat #4745 mouse IgG1, #4743 mouse IgG2a or #4744 mouse IgG2b).

It is strongly recommended to run both a positive and negative control alongside test antibodies and cell lines. CD71 (anti transferrin receptor) marker is recommended as a positive control for the mouse Fab IgG1 or 2a. Isotype matched IgG are recommended as negative controls. • Anti-CD71, clone MEM-189, IgG1 e.g. Sigma SAB4700520100UG • Anti-CD71, clone CYG4, IgG2a e.g. Biologend 334102 • Isotype controls, depending on isotype being studied – Mouse IgG1, e.g. R&D Systems, MAB002 or Biolegend 400124 – Mouse IgG2a e.g. Biolegend 401501 – Mouse IgG2b e.g. Biolegend 400322

• Test antibody of interest (at known concentration) containing mouse Fc region. – Recommend using Azide-free antibodies when available. • Target cells of interest. • Target cell growth media • Effector cell culture media • PBS (w/o Ca2+/Mg2+, Life Tech 14190). • 96-well flat bottom microplate (e.g. Corning® 3595) for imaging • 96-well round round bottom plate (e.g. Corning® 3799) or amber microtube (e.g. Cole parmer® MCT-150-X) for conjugation step. Additional material for non-adherent cell types • Poly-L-ornithine, PLO (Sigma P4957)

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IncuCyte® Live-cell Immunocytochemistry

Quick guide 1 Seed cells

Cell seeding Seed cells (50 µl/well, 5-30K/well) into 96-well plate. NOTE: for non-adherent cell types, PLO coat plate prior to cell seeding.

2 2 Label test antibody

3 Add Incucyte Opti-Green

4 Add labeled AB

IncuCyte® OptiGreen background suppressor addition Add 50 µl/well, (3x final concentration).

IncuCyte® FabFluor-488labeled antibody addition Add antibody-FabFluor mix (50 µl/well) to cell plate. Non-adherent cells – spin plate

Labeling of test antibody NOTE: It is recommend to use low azide or azide-free antibodies (e.g. LEAF™ from Biolegend). Effects on cell growth from high concentrations of azide have been observed in some cell types. If this is of concern, the buffer can be exchanged using a simple desalting column (e.g. Zeba from Thermo Scientific). 2.1. Rehydrate IncuCyte® FabFluor-488 Antibody Labeling reagent with 100 µL sterile water (final concentration = 0.5 mg/mL). NOTE: A 1:3 molar ratio of test antibody to IncuCyte® FabFluor-488 reagent is recommended. The labeling reagent is a third of the size of a standard antibody. Therefore, equal mg/mL quantities produce a 1:3 molar ratio of test antibody to labeling Fab.

5 Live-cell Fluorescent imaging

Automated imaging and quantitative analysis Capture images, (time span and objective depends on assay and cells type, 10x or 20x) in IncuCyte.

2.2. Mix test antibody with dilute IncuCyte® FabFluor-488 Antibody Labeling reagent and target cell growth media in a round bottom microplate or amber tube to protect from light. Prepare sufficient quantity to enable 50 µL/well at 3X final assay concentration. NOTE: WE strongly recommend using both a negative and positive control antibody (see Recommended Materials above) a. Add test antibody at 3X the final antibody concentration. Recommendation: A final concentration of <1.5 µg/mL of test antibody. A reasonable starting concentration is 1 µg/ mL (e.g. 3X working concentration = 3 µg/mL). b. Add IncuCyte® FabFluor-488 Antibody Labeling reagent at a 1:3 (test antibody:FabFluor) molar ratio. See Example calculations below. c. Add media to dilute to 3X final assay concentration. Triturate to mix. d. Incubate for 15 minutes at 37°C in the dark to allow for conjugation.

NOTE: Reagent is light sensitive, keep in amber or foil wrapped tubes. Remaining re-hydrated reagent can be aliquoted and stored at -80°C (avoid freezing and thawing, stable for > year).

IncuCyte® Live-cell Immunocytochemistry Assay Methodology 1

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1a - Seed target cells of interest - Adherent cell 1.1. Harvest cells of interest and determine cell concentration (e.g. trypan blue + hemocytometer). 1.2. Prepare cell seeding stock in target cell growth media to achieve 40-50% confluence after 2-6 h. Suggested starting range 5,000-20,000 cells/well (depends on cell type used). NOTE: Seeding density must be optimized for each cell type. 1.3. Using a multi-channel pipette, seed cells (50 μL per well) into a 96-well flat bottom microplate. Lightly tap plate side to ensure even liquid distribution in well. 1.4. Remove bubbles from all wells by gently squeezing a wash bottle (containing 70-100% ethanol with the inner straw removed) to blow vapor over the surface of each well. 1.5. Allow cells to settle on a level surface for 30 minutes at room temperature, then place in IncuCyte® live-cell analysis system to monitor cell confluence. NOTE: Depending on cell type, plates can be used in assay once cells have adhered to plastic and achieved normal cell morphology e.g. 2-3 hr for HT1080. Some cell types may require overnight incubation.

1b - Non-adherent cells NOTE: For this assay, non-adherent cells will be the last addition to the plate (prepare suspension during the antibody conjugation step). 1.1.Coat a 96-well flat bottom plate with relevant coating matrix. We recommend coating with 50 µL of either 0.01% poly-L-ornithine solution (Sigma P4957) or 5 µg/ mL fibronectin (Sigma P4957) diluted in 0.1% BSA. Coat for 1 hour at ambient temperature, remove solution from wells, and then allow plates to dry for 30-60 minutes prior to cell addition. NOTE: Some optimization of plate coatings may be required. 1.2.Count cells of interest and determine cell concentration (e.g. trypan blue + hemocytometer). 1.3. Prepare cell seeding stock in target cell growth media, suggest starting range of 20,000 – 40,000 cells/well in 50 µL (depends on cell type used). 1.4. Using a multi-channel pipette, seed cells (50 μL per well) into a 96-well flat bottom microplate. 1.5. Remove bubbles from all wells by gently squeezing a wash bottle (containing 70-100% ethanol with the inner straw removed) to blow vapor over the surface of each well. 1.6. Allow cells to settle on a level surface for 30 minutes at room temperature then place in IncuCyte® live-cell analysis system to monitor cell confluence. NOTE: To reduce settling time cell plates can be centrifuged for 1 minute at 50 g.

Example calculations of antibody labeling using positive control anit-CD71 at 1 mg/mL stock concentration

1. Required final assay concentration of test antibody — 1 μg/mL for anti-CD71 is recommended for positive control wells. Working concentration = 3X, or 3 μg/mL.

b. Determine volume of IncuCyte FabFluor:

2. Determine volume of labeled antibody required at 3X final assay concentration (i.e. dilution of 1:3 recommended upon addition to cells): [# wells] x 50 μL (plus additional required to prepare dilution series if desired). E.g. For 8 replicates of 1:3 dilution of labeled test antibody: 8 x 50 μL= 400 μL minimum (500μL used for this example) 3. Calculate volumes of test antibody, IncuCyte FabFluor reagent, and media required to provide 3X final assay concentration of labeled test antibody. a. Determine volume of test antibody:

[Total volume] μL x [Working concentration test antibody] μg/mL / [Stock concentration test antibody] mg/ mL /1000

[Volume of test antibody] μL X [Stock concentration of test antibody] mg/mL / [Stock concentration of FabFluor] mg/mL 1.5 μL x 1 mg/mL / 0.5 mg/mL = 3.0 μL

NOTE: IncuCyte FabFluor reagent is a third of the molecular weight of a standard antibody. Therefore, equal volumes of equal mg/mL quantities produce a 1:3 molar ratio of test antibody to FabFluor as MW of a typical antibody is ~3x of FabFluor. In this case, the stock concentration in mg/mL of test antibody is twice that of FabFluor. Therefore, 2X volume of FabFluor is required. c. Determine volume of media: [Total volume] – [Test antibody volume] – [FabFluor volume]

500 μL – 1.5 µL – 3.0 μL = 495.5 μL

500 μL x 3 μg/mL / 1 mg/mL / 1000 = 1.5 μL

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IncuCyte® Live-cell Immunocytochemistry

Dilution of IncuCyte Opti-Green background suppressor 3.1. Dilute Opti-Green stock in complete growth media for a final assay concentration of 0.5 mM or 1:200 dilution of stock (see calculations below). NOTE: A final assay concentration of 0.5 mM has proven to be suitable across a range of cell types, however some optimization may be required to assess cell proliferation in the presence of Opti-Green. For lower expressed markers, Opti-green may be increased

in some cell types (1 mM or 1:100 dilution of stock final assay concentration), allowing for a higher concentration of test antibody to be added (<3 µg/mL). A full list of pre-assessed cell types is included at the end of this protocol.

Example calculations for Opti-Green background suppressor 1. Required final assay concentration of Opti-Green Background Suppressor — 0.5 mM. Working concentration = 3X, or 1.5 mM. 2. Determine volume of Opti-Green Background Suppressor required at 3X final assay concentration (i.e. dilution of 1:3 recommended upon addition to cells): [# wells] x 50 μL (plus additional required to prepare dilution series if desired). E.g. For 96 replicates of 1:3 dilution of Opti-Green Background Suppressor:

3. Calculate volume of Opti-Green Background Suppressor required to provide 3X final assay concentration. a. Determine volume of Opti-Green Background Suppressor: [Total volume] μL x [Working concentration Opti-Green Background Suppressor] mM / [Stock concentration Opti-Green Background Suppressor] mM 5000 μL x 1.5 mM / 100 mM = 75 μL b. Determine volume of media:[Total volume] – [OptiGreen Background Suppressor]

5000 μL – 75 μL = 4925 μL

96 x 50 μL= 4800 μL minimum (5000 μL used for this example)

Add IncuCyte FabFlour-488 test antibody and Opti-Green to Cells Adherent cells 4.1 Remove cell plate from incubator. 4.2 Using a multi-channel pipette: a. Add 50 μL of diluted Opti-Green to wells b. Add 50 μL of labeled antibody to required test wells. c. Remove any bubbles and place plate in IncuCyte® live-cell analysis system. 4.3 Place plate in IncuCyte® Live-cell analysis system.

Non-adherent cells 4.1 Add reagents to matrix coated plate: a. Add 50 μL of diluted Opti-green b. Add 50 μL of labelled antibody to required test wells. c. Add 50 μL of cell suspension to wells. d. Remove any bubbles 4.2 Allow the plate to sit for 30 minutes at room temperature to allow even settling, or centrifuge at 50 g for 1 minute. 4.3 Place plate in IncuCyte® Live-cell analysis system.

Aquire images and analyze 5.1 Using IncuCyte® software, schedule 24-hour repeat scanning for every 2-3 h. a. Scan on schedule, Standard. b. Channel selection: select “phase” and “green” c. Objective: 10x or 20x depending on cell types used. Generally, 10x is recommended for adherent cells, and 20x for non-adherent or smaller cells. 5.2 To generate the metrics, user must create an Analysis Definition suited to the cell type, assay conditions and magnification selected. 5.3 Select images from a well containing a positive signal and an isotype control well (negative signal) at a time point where staining is visible. 5.4 In the Analysis Definition: a. Set mask for phase confluence measure with green channel turned off. b. Turn green channel on and mask green objects. Exclude background fluorescence using the background subtraction feature. The feature “TopHat” will subtract local background from brightly fluorescent objects within a given radius; applicable

Analysis Guidelines Staining of surfaced expressed protein will appear as a green ring followed by intracellular green signal as there will be internalization of the signal over time (time depends on cell type studied). Suggested metrics for data analysis are shown below: 1. Quantification of fluorescence area (“total object area” or “green object confluence”). Suggested metric: Analyze using “Total green Object Area (μm2/well)”.

2. Quantification of intensity, integrated over the area of detectable fluorescence (i.e. “Total Integrated Intensity”). Suggested metric: Analyze using “Total green Object Integrated Intensity (GCU x μm2/ well)” metrics. 3. To correct for cell proliferation, it is advisable to normalize the area measurement for cell coverage (e.g. “green object confluence”/”phase confluence”).

Table 1. IncuCyte® Opti-Green concentration recommendations Cell Type

Adherent

Nonadherent

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for analyzing objects which change in fluorescence intensity over time. i. The radius chosen should reflect the size of fluorescent objects but contain enough background to reliably estimate background fluorescence in the image; 20-30 μm is often a useful starting point. ii. The threshold chosen will ensure that objects below a fluorescence threshold will not be masked. iii. Choose a threshold in which green objects are masked in the positive response image but low numbers in the isotype control, negative response well. NOTE: For non-adherent cell types, with images captured using a 20x objective, analysis can be performed using the IncuCyte® S3 Cell-by-Cell analysis Module (PN 9600-0031). This enables individual cell identification and subsequent classification into subpopulations based on properties including fluorescence intensity, size and shape. For further details of this analysis module and it’s applications see: www.essenbioscience.com/cell-by-cell

Cell Line

Type

HT1080 NR

Epithelial (fibrosarcoma)

A549 NR

Lung

MDA-MB-231 NR

Breast

MCF-7 NR

Breast

SKOV-3 NR

Ovary

Jurkat

T cell leukemia

CCRF-CEM

T cell leukemia

Raji

B cell lymphoma

Ramos

B cell lymphoma

THP-1

Monocytic

HL-60

Pre-myelocytic

PBMCs

Peripheral blood cells

Concentration Range Tested

Recommended Concentration

0.25mM-30mM

1mM

0.125mM-10mM

0.5mM

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IncuCyte® S3 Multi-Spheroid Assay

Quick guide 1

COAT PLATE (Day 0)

Coat plate (50% Matrigel®, 40 µL/well). Polymerize at 37°C for 30 minutes.

This protocol describes a solution for creating multi-spheroids using 96-well flat bottom plates coated with Matrigel®. The method utilizes the IncuCyte® live-cell analysis system for imagebased brightfield and fluorescence measurements of multispheroid size (area), number and health.

Recommended materials

• 96-well flat bottom TC-treated microplate (Corning Cat #3595)

• IncuCyte® Cool Accessories (Cat # 1500-0078) - CoolBox 96F System (Includes x1 Block with gelpack and Coolsink 96F)

• Wet ice • Serum-free cell culture media for Matrigel® dilutions • Complete culture media for cell culture and assay

• IncuCyte® CytoLight Red or Green Lentivirus Reagent (Essen Bioscience, Cat #4481 or #4482)

• IncuCyte® S3 Spheroid Software Module, version 2018A (Essen Cat #9600-0019)

General Guidelines

• Follow manufacture’s guidelines for thawing and storing of 100% Matrigel®. Thaw Corning® Matrigel® overnight by submerging the vial in ice cold water in the rear of a refrigerator (+ 4°C). Do not allow Matrigel® to warm to room temperature at any time as this will induce polymerization. • We recommend sourcing a batch of Matrigel® with a concentration of ≥ 8 mg/mL.

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Add cells (150 µL/well) and allow multi-spheroids to form (3 days).

IMPORTANT: 1. In advance of multi-spheroid experiments, it is important to have stored the Cool pack accessories at the correct temperatures for at least 4h: a. Coolbox x 1 (block with gelpack: -20°C), b. Coolsink 96F x1 (4°C)

• IncuCyte® NucLight Red or Green Lentivirus Reagent (Essen Bioscience, Cat #4476 or #4475)

• Manual multi-channel pipette

• All materials (e.g. culture-ware, reagents) that will come in contact with Matrigel® must be kept cold (on ice, stored at + 4°C).

ADD CELLS (Day 0)

MONITOR FORMATION (Day 0—3)

Place inside the IncuCyte and scan every six hours to monitor multi-spheroid formation.

ADD TREATMENTS (Day 3)

Add appropriate treatments (50 µL/well) at 4x final assay concentration.

Protocol

Required materials

• Matrigel®, protein concentration ≥ 8 mg/mL (Corning Cat #356234)

• Following Matrigel® coating, cell seeding and after treatment addition, remove bubbles from all wells by gently squeezing a wash bottle containing 70 – 100% ethanol, with the inner straw removed, to blow vapor over the surface of each well. • After placing the plate in the IncuCyte® live-cell analysis system, allow the plate to warm to 37°C for 30 minutes prior to scanning.

2. Keep all culture-ware and reagents coming in contact with Matrigel® on ice during the entire process. 3. Store pipette tips used for dispensing diluted Matrigel® at + 4°C.

Day 0 Coat plate with Matrigel® 1.1 In a cell culture hood, chill plates (10 – 15 minutes) on a pre-chilled Coolsink 96F within a Coolbox 96F box. 1.2 In a cold polypropylene tube, dilute 100% Matrigel® 1:1 in cold serum-free culture media (keep all Matrigel® solutions on ice). NOTE: To prevent incomplete gel formation, for coating we recommend using ≥ 4 mg/mL Matrigel®. a. To coat a single 96-well plate, add 2.5 mL of cold serumfree culture media to a pre-chilled polypropylene tube. b. Using a cold serological pipette, slowly pipette 2.5 mL of 100% Matrigel® into the serum-free media and, taking care to avoid bubbles, slowly mix by pipetting the solution up and down. 1.3 Pour prepared solution into a pre-chilled sterile reagent reservoir (keep on ice). 1.4 Using pre-chilled pipette tips and reverse pipetting technique, coat the pre-chilled 96-well plate by carefully adding 40 µL of diluted Matrigel® into the center of each well. a. While the plate is cold and Matrigel® is still liquid, gently rock the plate once within the Coolbox to ensure even coating of each well. NOTE: To avoid cell penetration to the base of the plate, coat wells with a minimum of 40 µL. Use of reverse pipetting technique is important to minimize bubbles.

1.5 Remove any bubbles using a wash bottle containing 70 – 100% ethanol, with the inner straw removed, to blow vapor over the surface of each well. 1.6 Place the plate in a 37°C incubator for 30 minutes to polymerize the Matrigel®. 2

Seed cells 2.1 Seed cells of interest (150 µL per well) at an appropriate density on top of polymerized Matrigel® base such that by day 3, multi-spheroids have formed with the desired size (e.g. 30 – 80 µm in diameter). NOTE: Seeding density will need to be optimized for each cell type used. As a guide, we recommend seeding A549, MCF-7 and MDA-MB-231 at 1000 - 2000 cells per well or SKOV-3 at 2000 - 4000 cells per well. 2.2 Place plate in a 37°C incubator for 30 minutes prior to scanning.

Day 0—3 3

Monitor multi-spheroid formation 3.1 Place the cell plate into the IncuCyte® live-cell analysis System and schedule 24 hour repeat scanning: a. Objective: 10x (96-well corning) 1 image per well b. Channel selection: Phase Contrast + Brightfield and “Red” or “Green” if required c. Scan type: Spheroid, Spheroid Type: Multi d. Scan interval: Every 6 hours

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Day 3 4

Add treatments 4.1 Once multi-spheroids have reached desired size, remove the plate from the IncuCyte and carefully add appropriate treatments at 4x final assay concentration (50 µL per well). 4.2. Continue to monitor multi-spheroid growth (e.g. every 6 hours for 7 days).

Re-feed cultures (optional) 5.1 Re-feed cultures every 3 – 4 days. Remove plate from IncuCyte. Carefully remove 100 µL of media per well and replace with 100 µL of media containing test agents (1x final assay concentration). 5.2 Return plate to the IncuCyte and continue to monitor multi-spheroid growth.

Analysis Guidelines Result: Size, number and viability/health measurements Suggested metric: Brightfield Object Area (Total, Average), Object Count Secondary metrics: Fluorescent metrics within a Brightfield Object Boundary

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IncuCyte® S3 Spheroid Viability Assay - Fluorescent Label For the quantification of fluorescently labeled spheroid growth and shrinkage.

This protocol describes a solution for creating single spheroids using a 96- or 384- well round-bottom, ultra-low attachment plate. This method utilizes the IncuCyte® live-cell analysis system for imagebased Brightfield and fluorescence within the Brightfield boundary of spheroid area measurements. Cell lines expressing fluorescent protein can be used to monitor spheroid health.

Required materials • IncuCyte® S3 Spheroid Software Module (Essen Cat # 96000019) • IncuCyte® S3 Spheroid software version 2017B Cell fluorescent label reagents and consumables

General Guidelines • Remove bubbles from all wells by gently squeezing a wash bottle containing 70-100% ethanol, with the inner straw removed, to blow vapor over the surface of each well. • After placing the plate in the IncuCyte® live-cell analysis system, allow the plate to warm to 37 °C for 30 minutes prior to scanning.

• IncuCyte® NucLight Red or Green BacMam 3.0 Reagent for nuclear labeling (Essen Cat # 4621 or 4622) • IncuCyte® NucLight Red or Green Lentivirus Reagent (EF-1 α, Puro) for nuclear labeling (Essen Cat # 4624 or 4625) • IncuCyte® CytoLight Red or Green Lentivirus Reagent (EF-1 α, Puro) for cytoplasmic labeling (Essen Cat # 4481 or 4482) • Matrigel® (Corning Cat#356234), optional • 96-well round-bottom, ultra-low attachment plate (e.g., Corning® Cat#7007, S-BIO Cat#MS-9096UZ, BRANDplates® Cat#7816 60, 7819 00, 7819 60) • 384-well round-bottom, ultra-low attachment plate (e.g., S-BIO Cat#MS-9384UZ) NOTE: Combination of cells expressing fluorescent proteins with cell health reagents (Cytotox, Annexin V) is NOT recommended.

Protocol 1

CELL SEEDING (Day 0)

Seed cells into 96W or 384W Ultra Low Attachment plate. Centrifuge.

SPHEROID FORMATION (Day 0–3)

Place plate inside the IncuCyte and scan every six hours.

ADD TREATMENTS Day 3

Add treatments to plate. Monitor spheroid growth and shrinkage.

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Live-Cell Analysis Handbook — Second Edition

Day 0 Seed cells 1.1. Seed cells of interest (100 µL per well for 96-well, 50 µL for 384-well) at an appropriate density into a 96- or 384-well ULA plate such that by day 3, spheroids have formed with the desired size (e.g., 200 – 500 µm after 3 days). Seeding density will need to be optimized for each cell line used, however, we recommend a range of 1,000 – 5,000 cells per well (10,000 – 50,000 cells per mL seeding stock). NOTE: Some cell lines may require the addition of a basement membrane extract, typically 2.5% v/v Matrigel®, to promote tight spheroid formation. 1.2. Centrifuge the ULA plate (125 g, 10 minutes) at room temperature (20-25°C).

Add treatments 3.1. Once spheroids have reached desired size (e.g., 200 – 500 µm), remove the ULA plate from the incubator and carefully add culture media supplemented with cell heath reagent (100 µL per well for 96-well, 25 µL per well for 384-well) containing test material (e.g. small molecules, antibodies; prepared at 2x final assay concentration for 96-well, 3x final assay concentration for 384-well). 3.2. Continue to monitor spheroid growth (e.g. every 6 h for 10 days). NOTE: It is not recommended to change media in this assay as it will disrupt spheroids containing necrosing or apoptotic cells.

Day 0—3 2

Spheroid formation 2.1. Place the cell plate into the IncuCyte live-cell analysis System and schedule 24 hour repeat scanning: a. Objective: 4x or 10x (96-well ULA) or 10x (384-well ULA), 1 image per well b. Channel selection: Phase Contrast; Brightfield; “Green” or “Red” if fluorescent label OR if a cell health reagent will be added post spheroid formation. c. Scan type: Spheroid. d. Scan interval: Every 6 hours.

Analysis Guidelines NOTE: Utilize the IncuCyte® S3 Spheroid Software module in the Brightfield channel to identify spheroid boundaries and analyze fluorescence as needed. See “Guidelines for Analysis,” which can be accessed from the IncuCyte® S3 Technical Notes folder as part of the GUI installer. 1. For parental (non-transduced) cells: Brightfield Boundary Measurements Result: Size of spheroid measurement Suggested Metric: Largest Brightfield object (avoid segmentation of small fragments) IncuCyte. Carefully remove 100 µL of media per well and replace with 100 µL of media containing test agents (1x final assay concentration).

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2. For cells expressing fluorescent protein: Fluorescent and Brightfield Boundary Measurements Result: Size and viability measurements Suggested Metric: Integrated intensity Secondary metric: Mean intensity

Essen BioScience, Inc., a Sartorius Company, advances live-cell research world wide with the IncuCyte® Live-Cell Analysis System. The IncuCyte system and suite of assays automatically collect and analyze images and data in real time—while cells remain undisturbed inside a standard incubator.

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© 2018 Essen BioScience. All rights reserved. IncuCyte, Essen BioScience and all names of Essen BioScience products are registered trademarks and the property of Essen BioScience unless otherwise specified. Essen Bioscience is a Sartorius Company. No part of this book may be reproduced in any form without permission in writing from Essen BioScience. Specifications subject to change without notice.

Live-Cell Analysis Handbook — 2nd Edition